Antibodies to tumor necrosis factor receptors 6α and 6β

ABSTRACT

The present invention relates to novel Tumor Necrosis Factor Receptor proteins. In particular, isolated nucleic acid molecules are provided encoding the human TNFR-6α &amp; -6β proteins. TNFR-6α &amp; -6β polypeptides and antibodies that bind TNFR-6α &amp; -6β polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of TNFR-6α &amp; -6β activity. Also provided are diagnostic methods for detecting immune system-related disorders and therapeutic methods for treating immune system-related disorders.

RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional patentapplication Ser. No. 09/935,727, filed Aug. 24, 2001, now U.S. Pat. No.7,285,267, which claims the benefit of priority under 35 U.S.C. § 119(e)based on U.S. Provisional Application Ser. Nos. 60/303,224 filed Jul. 6,2001; 60/252,131 filed Nov. 21, 2000; and 60/227,598 filed Aug. 25,2000. U.S. Non-Provisional patent application Ser. No. 09/935,727, filedAug. 24, 2001 is also a continuation-in-part of U.S. Non-Provisionalpatent application Ser. No. 09/518,931 filed Mar. 3, 2000 , now U.S.Pat. No. 7,186,800, which claims the benefit of priority under 35 U.S.C.§ 119(e) based on U.S. Provisional Application Ser. No. 60/168,235 filedDec. 1, 1999; 60/146,371 filed Aug. 2, 1999; 60/131,964 filed Apr. 30,1999; 60/131,279 filed Apr. 27, 1999; 60/124,092 filed Mar. 12, 1999;and 60/121,774 filed Mar. 4, 1999. U.S. Non-Provisional patentapplication Ser. No. 09/518,931 is also a continuation-in-part of U.S.Non-Provisional patent application Ser. No. 09/006,352 filed Jan. 13,1998 which claims the benefit of priority under 35 U.S.C. § 119(e) basedon U.S. Provisional Application Ser. No. 60/035,496 filed Jan. 14, 1997.U.S. Non-Provisional patent application Ser. No. 09/935,727, filed Aug.24, 2001 is also a continuation-in-part of U.S. Non-Provisionalapplication Ser. No. 09/006,352 filed Jan. 13, 1998. This application isalso a continuation-in-part of U.S. Non-Provisional patent applicationSer. No. 09/518,931 filed Mar. 3, 2000 now U.S. Pat. No. 7,186,800. Thisapplication is also a continuation-in-part of U.S. Non-Provisionalapplication Ser. No. 09/006,352 filed Jan. 13, 1998. Each of the aboveU.S. Provisional and Non-Provisional patent applications is herebyincorporated by reference in its entirety. Each of the above U.S.Provisional and Non-Provisional patent applications is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel human genes encoding polypeptideswhich are members of the TNF receptor family. More specifically,isolated nucleic acid molecules are provided encoding human polypeptidesnamed tumor necrosis factor receptor-6α & -6β hereinafter sometimesreferred to as “TNFR-6α, & TNFR-6β” or generically as “TNFRpolypeptides”. TNFR polypeptides are also provided, as are vectors, hostcells and recombinant methods for producing the same. The inventionfurther relates to screening methods for identifying agonists andantagonists of TNFR polypeptide activity. Also provided are diagnosticand therapeutic methods utilizing such compositions.

BACKGROUND OF THE INVENTION

Many biological actions, for instance, response to certain stimuli andnatural biological processes, are controlled by factors, such ascytokines. Many cytokines act through receptors by engaging the receptorand producing an intra-cellular response.

For example, tumor necrosis factors (TNF) alpha and beta are cytokineswhich act through TNF receptors to regulate numerous biologicalprocesses, including protection against infection and induction of shockand inflammatory disease. The TNF molecules belong to the “TNF-ligand”superfamily, and act together with their receptors or counter-ligands,the “TNF-receptor” superfamily. So far, sixteen members of the TNFligand superfamily have been identified and seventeen members of theTNF-receptor superfamily have been characterized.

Among the ligands there are included TNF-α, lymphotoxin-α (LT-α, alsoknown as TNF-β), LT-β (found in complex heterotrimer LT-α2-β), FasL,CD40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF). Thesuperfamily of TNF receptors includes the p55TNF receptor, p75TNFreceptor, TNF receptor-related protein, FAS antigen or APO-1, CD40,CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor (Meager, A.,Biologicals, 22:291-295 (1994)).

Many members of the TNF-ligand superfamily are expressed by activatedT-cells, implying that they are necessary for T-cell interactions withother cell types which underlie cell ontogeny and functions. (Meager,A., supra).

Considerable insight into the essential functions of several members ofthe TNF receptor family has been gained from the identification andcreation of mutants that abolish the expression of these proteins. Forexample, naturally occurring mutations in the FAS antigen and its ligandcause lymphoproliferative disease (Watanabe-Fukunaga, R., et al., Nature356:314 (1992)), perhaps reflecting a failure of programmed cell death.Mutations of the CD40 ligand cause an X-linked immunodeficiency statecharacterized by high levels of immunoglobulin M and low levels ofimmunoglobulin G in plasma, indicating faulty T-cell-dependent B-cellactivation (Allen, R. C. et al., Science 259:990 (1993)). Targetedmutations of the low affinity nerve growth factor receptor cause adisorder characterized by faulty sensory innovation of peripheralstructures (Lee, K. F. et al., Cell 69:737 (1992)).

TNF and LT-α are capable of binding to two TNF receptors (the 55- and75-kd TNF receptors). A large number of biological effects elicited byTNF and LT-α, acting through their receptors, include hemorrhagicnecrosis of transplanted tumors, cytotoxicity, a role in endotoxicshock, inflammation, immunoregulation, proliferation and anti-viralresponses, as well as protection against the deleterious effects ofionizing radiation. TNF and LT-α are involved in the pathogenesis of awide range of diseases, including endotoxic shock, cerebral malaria,tumors, autoimmune disease, AIDS and graft-host rejection (Beutler, B.and Von Huffel, C., Science 264:667-668 (1994)). Mutations in the p55Receptor cause increased susceptibility to microbial infection.

Moreover, an about 80 amino acid domain near the C-terminus of TNFR1(p55) and Fas was reported as the “death domain,” which is responsiblefor transducing signals for programmed cell death (Tartaglia et al.,Cell 74:845 (1993)). Apoptosis, or programmed cell death, is aphysiologic process essential to the normal development and homeostasisof multicellular organisms (H. Steller, Science 267, 1445-1449 (1995)).Derangements of apoptosis contribute to the pathogenesis of severalhuman diseases including cancer, neurodegenerative disorders, andacquired immune deficiency syndrome (C. B. Thompson, Science 267,1456-1462 (1995)). Recently, much attention has focused on the signaltransduction and biological function of two cell surface deathreceptors, Fas/APO-1 and TNFR-1 (J. L. Cleveland, J. N. Ihle, Cell 81,479-482 (1995); A. Fraser, G. Evan, Cell 85, 781-784 (1996); S. Nagata,P. Golstein, Science 267, 1449-56 (1995)). Both are members of the TNFreceptor family which also include TNFR-2, low affinity NGFR, CD40, andCD30, among others (C. A. Smith, et al., Science 248, 1019-23 (1990); M.Tewari, V. M. Dixit, in Modular Texts in Molecular and Cell Biology M.Purton, Heldin, Carl, Ed. (Chapman and Hall, London, 1995). While familymembers are defined by the presence of cysteine-rich repeats in theirextracellular domains, Fas/APO-1 and TNFR-1 also share a region ofintracellular homology, appropriately designated the “death domain”,which is distantly related to the Drosophila suicide gene, reaper (P.Golstein, D. Marguet, V. Depraetere, Cell 81, 185-6 (1995); K. White etal., Science 264, 677-83 (1994)). This shared death domain suggests thatboth receptors interact with a related set of signal transducingmolecules that, until recently, remained unidentified. Activation ofFas/APO-1 recruits the death domain-containing adapter moleculeFADD/MORT1 (A. M. Chinnaiyan, K. O'Rourke, M. Tewari, V. M. Dixit, Cell81, 505-12 (1995); M. P. Boldin, et al., J. Biol Chem 270, 7795-8(1995); F. C. Kischkel, et al., EMBO 14, 5579-5588 (1995)), which inturn binds and presumably activates FLICE/MACH1, a member of theICE/CED-3 family of pro-apoptotic proteases (M. Muzio et al., Cell 85,817-827 (1996); M. P. Boldin, T. M. Goncharov, Y. V. Goltsev, D.Wallach, Cell 85, 803-815 (1996)). While the central role of Fas/APO-1is to trigger cell death, TNFR-1 can signal an array of diversebiological activities-many of which stem from its ability to activateNF-kB (L. A. Tartaglia, D. V. Goeddel, Immunol Today 13, 151-3 (1992)).Accordingly, TNFR-1 recruits the multivalent adapter molecule TRADD,which like FADD, also contains a death domain (H. Hsu, J. Xiong, D. V.Goeddel, Cell 81, 495-504 (1995); H. Hsu, H.-B. Shu, M.-P. Pan, D. V.Goeddel, Cell 84, 299-308 (1996)). Through its associations with anumber of signaling molecules including FADD, TRAF2, and RIP, TRADD cansignal both apoptosis and NF-kB activation (H. Hsu, H.-B. Shu, M.-P.Pan, D. V. Goeddel, Cell 84, 299-308 (1996); H. Hsu, J. Huang, H.-B.Shu, V. Baichwal, D. V. Goeddel, Immunity 4, 387-396 (1996)).

The effects of TNF family ligands and TNF family receptors are variedand influence numerous functions, both normal and abnormal, in thebiological processes of the mammalian system. There is a clear need,therefore, for identification and characterization of such receptors andligands that influence biological activity, both normally and in diseasestates. In particular, there is a need to isolate and characterize novelmembers of the TNF receptor family.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising, or alternatively consisting of, a polynucleotide encoding atleast a portion of a TNFR (i.e., TNFR-6α or TNFR-6β polypeptide) havingthe complete amino acid sequences shown in SEQ ID NOS:2 and 4,respectively, or the complete amino acid sequence encoded by a cDNAclone deposited as plasmid DNA as ATCC Deposit Number 97810 and 97809,respectively. The nucleotide sequence determined by sequencing thedeposited TNFR-6 alpha and TNFR-6 beta clones, which are shown in FIGS.1 and 2A-B (SEQ ID NOS:1 and 3, respectively), contain open readingframes encoding complete polypeptides of 300 and 170 amino acidresidues, respectively, including an initiation codon encoding anN-terminal methionine at nucleotide positions 25-27 and 73-75 in SEQ IDNOS: 1 and 3, respectively.

The TNFR proteins of the present invention share sequence homology withother TNF receptors. Splice variants TNFR-6 alpha and TNFR-6 beta showthe highest degree of sequence homology with the translation products ofthe human mRNAs for TNFR-I and -II (FIGS. 3A-P) (SEQ ID NOS:5 and 6,respectively) also including multiple conserved cysteine rich domains.

The TNFR-6 alpha and TNFR-6 beta polypeptides have predicted leadersequences of 30 amino acids each; and the amino acid sequence of thepredicted mature TNFR-6 alpha and TNFR-6 beta polypeptides are alsoshown in FIGS. 1 and 2A-B as amino acid residues 31-300 (SEQ ID NO:2)and 31-170 (SEQ ID NO:4), respectively.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a TNFR polypeptide having the completeamino acid sequence in SEQ ID NO:2 or 4, or as encoded by the cDNA clonecontained in ATCC Deposit No. 97810 or 97809; (b) a nucleotide sequenceencoding a mature TNFR polypeptide having the amino acid sequence atpositions 31-300 in SEQ ID NO:2, or 31-170 in SEQ ID NO:4, or as encodedby the cDNA clone contained in ATCC Deposit No. 97810 or 97809; (c) anucleotide sequence encoding a soluble extracellular domain of a TNFRpolypeptide having the amino acid sequence at positions 31 to 283 in SEQID NO:2 or 31 to 166 in SEQ ID NO:4, or as encoded by the cDNA clonecontained in the ATCC Deposit No. 97810 or 97809; (d) a nucleotidesequence encoding a fragment of a TNFR polypeptide having the amino acidsequence at positions 31 to 283 in SEQ ID NO:2 or 31 to 166 in SEQ IDNO:4, or as encoded by the cDNA clone contained in the ATCC Deposit No.97810 or 97809 wherein said fragment has TNFR-6α and/or TNFR-6βfunctional activity; and (e) a nucleotide sequence complementary to anyof the nucleotide sequences in (a), (b), (c), or (d) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise, or alternatively consist of, a polynucleotidehaving a nucleotide sequence at least 90% identical, and more preferablyat least 80%, 85%, 90%, 92%, or 95%, 96%, 97%, 98% or 99% identical, toany of the nucleotide sequences in (a), (b), (c), (d) and (e) above, ora polynucleotide which hybridizes under stringent hybridizationconditions to a polynucleotide in (a), (b), (c), (d), or (e) above. Thispolynucleotide which hybridizes does not hybridize under stringenthybridization conditions to a polynucleotide having a nucleotidesequence consisting of only A residues or of only T residues. Anadditional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising, or alternatively consistingof, a polynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a TNFR polypeptide having an amino acidsequence in (a), (b), (c), or (d) above.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofTNFR polypeptides or peptides by recombinant techniques.

The invention further provides an isolated TNFR polypeptide comprisingan amino acid sequence selected from the group consisting of: (a) theamino acid sequence of a full -length TNFR polypeptide having thecomplete amino acid sequence shown in SEQ ID NO:2 or 4 or as encoded bythe cDNA clone contained in ATCC Deposit No. 97810 or 97809; (b) theamino acid sequence of a mature TNFR polypeptide having the amino acidsequence at positions 31-300 in SEQ ID NO:2, or 31-170 in SEQ ID NO:4,or as encoded by the cDNA clone contained in ATCC Deposit No. 97810 or97809; (c) the amino acid sequence of a soluble extracellular domain ofa TNFR polypeptide having the amino acid sequence at positions 31 to 283in SEQ ID NO:2 or 31 to 166 in SEQ ID NO:4, or as encoded by the cDNAclone contained in ATCC Deposit No. 97810 or 97809; or (d) the aminoacid sequence of a fragment of the TNFR polypeptide having the aminoacid sequence at positions 31 to 283 in SEQ ID NO:2 or 31 to 166 in SEQID NO:4, or as encoded by the cDNA clone contained in ATCC Deposit No.97810 or 97809, wherein said fragment has TNFR-6αand/or TNFR-6βfunctional activity.

The polypeptides of the present invention also include polypeptideshaving an amino acid sequence at least 80% identical, more preferably atleast 85% identical, and still more preferably 90%, 92%, 95%, 96%, 97%,98% or 99% identical to those described in (a), (b), (c) or (d) above,as well as polypeptides having an amino acid sequence with at least 90%similarity, and more preferably at least 80%, 85%, 90%, 92%, or 95%similarity, to those above.

An additional embodiment of this aspect of the invention relates to apeptide or polypeptide which comprises the amino acid sequence of anepitope-bearing portion of a TNFR polypeptide having an amino acidsequence described in (a), (b), (c) or (d), above. Peptides orpolypeptides having the amino acid sequence of an epitope-bearingportion of a TNFR polypeptide of the invention include portions of suchpolypeptides with at least six or seven, preferably at least nine, andmore preferably at least about 30 amino acids to about 50 amino acids,although epitope-bearing polypeptides of any length up to and includingthe entire amino acid sequence of a polypeptide of the inventiondescribed above also are included in the invention.

In another embodiment, the invention provides an isolated antibody thatbinds specifically to a TNFR polypeptide having an amino acid sequencedescribed in (a), (b), (c) or (d) above. The invention further providesmethods for isolating antibodies that bind specifically to a TNFRpolypeptide having an amino acid sequence as described herein. Suchantibodies are useful diagnostically or therapeutically as describedbelow.

Tumor Necrosis Factor (TNF) family ligands are known to be among themost pleiotropic cytokines, inducing a large number of cellularresponses, including cytotoxicity, anti-viral activity, immunoregulatoryactivities, and the transcriptional regulation of several genes. Theinvention also provides for pharmaceutical compositions comprising TNFRpolypeptides, particularly human TNFR polypeptides, which may beemployed, for instance, to treat infectious disease including HIVinfection, endotoxic shock, cancer, autoimmune diseases, graft vs. hostdisease, acute graft rejection, chronic graft rejection,neurodegenerative disorders, myelodysplastic syndromes, ischemic injury(e.g., ischemic cardiac injury), toxin-induced liver disease, septicshock, cachexia and anorexia. Methods of treating individuals in need ofTNFR polypeptides are also provided.

The invention further provides compositions comprising a TNFRpolynucleotide or a TNFR polypeptide for administration to cells invitro, to cells ex vivo and to cells in vivo, or to a multicellularorganism. In certain particularly preferred embodiments of this aspectof the invention, the compositions comprise a TNFR polynucleotide forexpression of a TNFR polypeptide in a host organism for treatment ofdisease. Particularly preferred in this regard is expression in a humanpatient for treatment of a dysfunction associated with aberrantendogenous activity of a TNFR polypeptide.

In another aspect, a screening assay for agonists and antagonists isprovided which involves determining the effect a candidate compound hason TNFR polypeptide binding to a TNF-family ligand. In particular, themethod involves contacting the TNF-family ligand with a TNFR polypeptideand a candidate compound and determining whether TNFR polypeptidebinding to the TNF-family ligand is increased or decreased due to thepresence of the candidate compound. In this assay, an increase inbinding of a TNFR polypeptide over the standard binding indicates thatthe candidate compound is an agonist of TNFR polypeptide bindingactivity and a decrease in TNFR polypeptide binding compared to thestandard indicates that the compound is an antagonist of TNFRpolypeptide binding activity.

TNFR-6 alpha and TNFR-6 beta are expressed in endothelial cells,keratinocytes, normal prostate and prostate tumor tissue. For a numberof disorders of these tissues or cells, particularly of the immunesystem, significantly higher or lower levels of TNFR gene expression maybe detected in certain tissues (e.g., cancerous tissues) or bodilyfluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid)taken from an individual having such a disorder, relative to a“standard” TNFR gene expression level, i.e., the TNFR expression levelin healthy tissue from an individual not having the immune systemdisorder. Thus, the invention provides a diagnostic method useful duringdiagnosis of such a disorder, which involves: (a) assaying TNFR geneexpression level in cells or body fluid of an individual; (b) comparingthe TNFR gene expression level with a standard TNFR gene expressionlevel, whereby an increase or decrease in the assayed TNFR geneexpression level compared to the standard expression level is indicativeof disorder in the immune system.

An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of TNFR polypeptideactivity in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of an isolatedTNFR polypeptide of the invention or an agonist thereof.

A still further aspect of the invention is related to a method fortreating an individual in need of a decreased level of TNFR polypeptideactivity in the body comprising, administering to such an individual acomposition comprising a therapeutically effective amount of a TNFRantagonist. Preferred antagonists for use in the present invention areTNFR-specific antibodies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide sequence (SEQ ID NO:1) and deduced aminoacid sequence (SEQ ID NO:2) of TNFR-6α. The initial 30 amino acids(underlined) are the putative leader sequence.

FIGS. 2A-B the nucleotide sequence (SEQ ID NO:3) and deduced amino acidsequence (SEQ ID NO:4) of TNFR-6β. The initial 30 amino acids(underlined) are the putative leader sequence.

FIGS. 3A-P show an alignment created by the Clustal method using theMegaline program in the DNAstar suite comparing the amino acid sequencesof TNFR-6α (“TNFR-6 alpha” (SEQ ID NO:2)), and TNFR-6β (“TNFR-6 beta”(SEQ ID NO:4)) with other TNF receptors, as follows: TNFR1 (SEQ IDNO:5); TNFR2 (SEQ ID NO:6); NGFR (SEQ ID NO:7); LTbR (SEQ ID NO:8); FAS(SEQ ID NO:9); CD27 (SEQ ID NO:10); CD30 (SEQ ID NO:11); CD40 (SEQ IDNO:12); 4-1BB (SEQ ID NO:13); OX40 (SEQ ID NO:14); VC22 (SEQ ID NO:15);and CRMB (SEQ ID NO:16).

FIGS. 4 and 5 show separate analyses of the TNFR-6 alpha and TNFR-6 betaamino acid sequences, respectively. Alpha, beta, turn and coil regions;hydrophilicity; amphipathic regions; flexible regions; antigenic indexand surface probability are shown, as predicted for the amino acidsequence of SEQ ID NO:2 and SEQ ID NO:4, respectively, using the defaultparameters of the recited computer programs. In the “AntigenicIndex—Jameson-Wolf” graph, which indicates the location of the highlyantigenic regions of TNFR-6α and TNFR-6β, i.e., regions from whichepitope-bearing peptides of the invention may be obtained. Antigenicregions of TNFR-6α, incude from about Ala-31 to about Thr-46, from aboutPhe-57 to about Thr-117, from about Cys-132 to about Thr-175, from aboutGly-185 to about Thr-194, from about Val-205 to about Asp-217, fromabout Pro-239 to about Leu-264, and from about Ala-283 to about Pro-298(SEQ ID NO:2). Antigenic regions of TNFR-6β, include from about Ala-31to about Thr-46, from about Phe-57 to about Gln-80, from about Glu-86 toabout His-106, from about Thr-108 to about Phe-119, from about His-129to about Val-138, and from about Gly-142 to about Pro-166 (SEQ ID NO:4).These polypeptide fragments have been determined to bear antigenicepitopes of the TNFR-6 alpha and TNFR-6 beta polypeptides by theanalysis of the Jameson-Wolf antigenic index.

The data presented in FIGS. 4 and 5 are also represented in tabular formin Tables I and II, respectively. The columns are labeled with theheadings “Res”, “Position”, and Roman Numerals I-XIV. The columnheadings refer to the following features of the amino acid sequencepresented in FIG. 4, (Table I) and FIG. 5 (Table II): “Res”: amino acidresidue of SEQ ID NO:2 (FIG. 1) or SEQ ID NO:4 (FIG. 2A); “Position”:position of the corresponding residue within of SEQ ID NO:2 (FIG. 1) orSEQ ID NO:4 (FIG. 2A); I: Alpha, Regions—Garnier-Robson; II: Alpha,Regions—Chou-Fasman; III: Beta, Regions—Garnier-Robson; IV: Beta,Regions—Chou-Fasman; V: Turn, Regions—Garnier-Robson; VI: Turn,Regions—Chou-Fasman; VII: Coil, Regions—Garnier-Robson; VIII:Hydrophilicity Plot—Kyte-Doolittle; IX: Hydrophobicity Plot—Hopp-Woods;X: Alpha, Amphipathic Regions—Eisenberg; XI: Beta, AmphipathicRegions—Eisenberg; XII: Flexible Regions—Karplus-Schulz; XIII: AntigenicIndex—Jameson-Wolf; and XIV: Surface Probability Plot—Emini.

FIG. 6 shows the nucleotide sequences of HELDI06R (SEQ ID NO:17) andHCEOW38R (SEQ ID NO:18) which are related to SEQ ID NOS:1 and 3.

FIGS. 7A-B show TNFR6 alpha blocking of Fas ligand mediated cell death.Jurkat T-cells were treated with a combination of Fas ligand and TNFR 6alpha Fc receptor for 16 hours. To measure the levels of viable cellsafter treatment, cells were incubated for 5 hours with 10% ALOMAR blueand examined spectrophotometrically at OD 570nm-630nm. All samples weretested in triplicate. TNFR6 alpha-Fc appears to block Fas ligandmediated apoptosis of Jurkat cells in a dose dependent manner aseffectively as Fas-Fc.

DETAILED DESCRIPTION

The present invention provides isolated nucleic acid moleculescomprising, or alternatively consisting of, a polynucleotide encoding aTNFR-6α or -6β polypeptide, generically “TNFR polypeptide(s)” having theamino acid sequence shown in SEQ ID NOS:2 and 4, respectively, whichwere determined by sequencing cloned cDNAs. The nucleotide sequencesshown in FIGS. 1 and 2A-B (SEQ ID NOS:1 and 3) were obtained bysequencing the HPHAE52 and HTPCH84 clones, respectively, which weredeposited on Nov. 22, 1996 at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209 and given accessionnumbers ATCC 97810 and 97809, respectively. The deposited clones arecontained in the pBluescript SK(−) plasmid (Stratagene, La Jolla,Calif.).

The TNFR-6 alpha and TNFR-6 beta proteins of the present invention aresplice variants which share an identical nucleotide and amino acidsequence over the N-terminal 142 residues of the respective proteins.The amino acid sequences of these proteins are about 23% similar to andshare multiple conserved cysteine rich domains with the translationproduct of the human TNFR-2 mRNA (FIGS. 3A-P) (SEQ ID NO:6).Importantly, these proteins share substantial sequence similarity over apolypeptide sequence including four repeated cysteine rich motifs withsignificant intersubunit homology. TNFR-2 is thought to exclusivelymediate human T-cell proliferation by TNF (PCT WO/94/09137).

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc., FosterCity, Calif.), and all amino acid sequences of polypeptides encoded byDNA molecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

By “nucleotide sequence” of a nucleic acid molecule or polynucleotide isintended, for a DNA molecule or polynucleotide, a sequence ofdeoxyribonucleotides, and for an RNA molecule or polynucleotide, thecorresponding sequence of ribonucleotides (A, G, C and U), where eachthymidine deoxyribonucleotide (T) in the specified deoxyribonucleotidesequence is replaced by the ribonucleotide uridine (U).

Using the information provided herein, such as the nucleotide sequencesin FIGS. 1 and 2A-B (SEQ ID NOS:1 and 3), a nucleic acid molecule of thepresent invention encoding a TNFR polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the TNFR-6α and TNFR-6β clones (FIGS. 1 and 2A-B, respectively) wereidentified in cDNA libraries from the following tissues: endothelialcells, keratinocytes, normal prostate tissue, and prostate tumor tissue.

The determined nucleotide sequences of the TNFR cDNAs of FIGS. 1 and2A-B (SEQ ID NOS:1 and 3) contain open reading frames encoding proteinsof 300 and 170 amino acid residues, with an initiation codon atnucleotide positions 25-27 and 73-75 of the nucleotide sequences inFIGS. 1 and 2A-B (SEQ ID NOS:1 and 3), respectively.

The open reading frames of the TNFR-6α and TNFR-6β genes share sequencehomology with the translation product of the human mRNA for TNFR-2,including the soluble extracellular domain of about residues 31-283 ofSEQ ID NO:2 and 31-166 of SEQ ID NO:4, respectively.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above, the actual complete TNFR polypeptidesencoded by the deposited cDNAs, which comprise about 300 and 170 aminoacids, may be somewhat longer or shorter. More generally, the actualopen reading frames may be anywhere in the range of ±20 amino acids,more likely in the range of ±10 amino acids, of that predicted from thefirst methionine codon from the N-terminus shown in FIGS. 1 and 2A-B(SEQ ID NOS:1 and 3), which is in-frame with the translated sequencesshown in each respective figure. It will further be appreciated that,depending on the analytical criteria used for identifying variousfunctional domains, the exact “address” of the extracellular andtransmembrane domain(s) of the TNFR polypeptides may differ slightlyfrom the predicted positions above. For example, the exact location ofthe extracellular domain or antigenic regions in SEQ ID NO:2 and SEQ IDNO:4 may vary slightly (e.g., the address may “shift” by about 1 toabout 20 residues, more likely about 1 to about 5 residues) depending onthe criteria used to define the domains and antigenic regions. In anyevent, as discussed further below, the invention further providespolypeptides having various residues deleted from the N-terminus of thecomplete polypeptide, including polypeptides lacking one or more aminoacids from the N-terminus of the extracellular domain described herein,which constitute soluble forms of the extracellular domains of theTNFR-6α and TNFR-6β proteins.

The amino acid sequences of the complete TNFR proteins include a leadersequence and a mature protein, as shown in SEQ ID NOS:2 and 4. More inparticular, the present invention provides nucleic acid moleculesencoding mature forms of the TNFR proteins. Thus, according to thesignal hypothesis, once export of the growing protein chain across therough endoplasmic reticulum has been initiated, proteins secreted bymammalian cells have a signal or secretory leader sequence which iscleaved from the complete polypeptide to produce a secreted “mature”form of the protein. Most mammalian cells and even insect cells cleavesecreted proteins with the same specificity. However, in some cases,cleavage of a secreted protein is not entirely uniform, which results intwo or more mature species of the protein. Further, it has long beenknown that the cleavage specificity of a secreted protein is ultimatelydetermined by the primary structure of the complete protein, that is, itis inherent in the amino acid sequence of the polypeptide. Therefore,the present invention provides a nucleotide sequence encoding a matureTNFR polypeptide having the amino acid sequence encoded by a cDNA cloneidentified as ATCC Deposit No. 97810 or 97809. By the “mature TNFRpolypeptides having the amino acid sequence encoded by a cDNA clonecontained in the plasmid deposited as ATCC Deposit No. 97810, or 97809”is meant the mature form(s) of the protein produced by expression in amammalian cell (e.g., COS cells, as described below) of the completeopen reading frame encoded by the human DNA sequence of the clonecontained in the deposited vector.

In addition, methods for predicting whether a protein has a secretoryleader as well as the cleavage point for that leader sequence areavailable. For instance, the method of McGeoch (Virus Res. 3:271-286(1985)) uses the information from a short N-terminal charged region anda subsequent uncharged region of the complete (uncleaved) protein. Themethod of von Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) uses theinformation from the residues surrounding the cleavage site, typicallyresidues −13 to +2 where +1 indicates the amino terminus of the matureprotein. The accuracy of predicting the cleavage points of knownmammalian secretory proteins for each of these methods is in the rangeof 75-80% (von Heinje, supra). However, the two methods do not alwaysproduce the same predicted cleavage point(s) for a given protein.

In the present case, the deduced amino acid sequence of the completeTNFR polypeptides were analyzed by a computer program “PSORT”, availablefrom Dr. Kenta Nakai of the Institute for Chemical Research, KyotoUniversity (see K. Nakai and M. Kanehisa, Genomics 14:897-911 (1992)),which is an expert system for predicting the cellular location of aprotein based on the amino acid sequence. As part of this computationalprediction of localization, the methods of McGeoch and von Heinje areincorporated. The analysis of the TNFR amino acid sequences by thisprogram provided the following results: TNFR-6α & TNFR-6β encode maturepolypeptides having the amino acid sequences of residues 31-300 and31-170 of SEQ ID NOS:2 and 4, respectively.

In certain preferred embodiments, TNFR-6α & TNFR-6β encode maturepolypeptides having the amino acid sequences of residues 31-299 and31-169 of SEQ ID NOS:2 and 4, respectively.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. However, a nucleic acid contained in a clone that is a memberof a mixed clone library (e.g., a genomic or cDNA library) and that hasnot been isolated from other clones of the library (e.g., in the form ofa homogeneous solution containing the clone without other members of thelibrary) or a chromosome isolated or removed from a cell or a celllysate (e.g., a “chromosome spread”, as in a karyotype), is not“isolated” for the purposes of this invention. As discussed furtherherein, isolated nucleic acid molecules according to the presentinvention may be produced naturally, recombinantly, or synthetically.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) with an initiationcodon at positions 25-27 and 73-75 of the nucleotide sequences shown inSEQ ID NOS:1 and 3, respectively.

Also included are DNA molecules comprising the coding sequence for thepredicted mature TNFR polypeptides shown at positions 31-300 and 31-170of SEQ ID NOS:2 and 4, respectively.

Also included are DNA molecules comprising the coding sequence for thepredicted mature TNFR polypeptides shown at positions 31-299 and 31-169of SEQ ID NOS:2 and 4, respectively.

In specific embodiments, the present invention encompasses isolatednucleic acid molecules comprising a polynucleotide sequence encodingexon 1 of TNFR-6 alpha, (i.e., a polynucleotide sequence comprisingnucleotides 1-424 of SEQ ID NO:28 which corresponds to nucleotides25-448 of SEQ ID NO:1). In other embodiments, the present inventionencompasses isolated nucleic acid molecules comprising a polynucleotidesequence encoding exon 2 of TNFR-6 alpha, (i.e., a polynucleotidesequence comprising nucleotides 561-755 of SEQ ID NO:28 whichcorresponds to nucleotides 449-643 of SEQ ID NO:1). In otherembodiments, the present invention encompasses isolated nucleic acidmolecules comprising a polynucleotide sequence encoding exon 3 of TNFR-6alpha, (i.e., a polynucleotide sequence comprising nucleotides 1513-1793of SEQ ID NO:28 which corresponds to nucleotides 644-924 of SEQ IDNO:1).

In still other embodiments, the present invention comprises isolatednucleic acid molecules comprising a polynucleotide sequence encodingexons 1 and 2 of TNFR-6 alpha. In other embodiments, the presentinvention comprises isolated nucleic acid molecules comprising apolynucleotide sequence encoding exons 1 and 3 of TNFR-6 alpha. In otherembodiments, the present invention comprises isolated nucleic acidmolecules comprising a polynucleotide sequence encoding exons 2 and 3 ofTNFR-6 alpha.

In addition, isolated nucleic acid molecules of the invention includeDNA molecules which comprise a sequence substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encode a TNFR protein. Of course, the genetic code andspecies-specific codon preferences are well known in the art. Thus, itwould be routine for one skilled in the art to generate the degeneratevariants described above, for instance, to optimize codon expression fora particular host (e.g., change codons in the human mRNA to thosepreferred by a bacterial host such as E. coli).

In another aspect, the invention provides isolated nucleic acidmolecules encoding a TNFR polypeptide having an amino acid sequenceencoded by the cDNA clone contained in the plasmid deposited as ATCCDeposit No. 97810 or 97809. Preferably, this nucleic acid molecule willencode the mature polypeptide encoded by the above-described depositedcDNA clone.

The invention further provides an isolated nucleic acid molecule havingthe nucleotide sequence shown in FIG. 1 or 2 (SEQ ID NO:1 or 3) or thenucleotide sequence of the TNFR cDNAs contained in the above-describeddeposited clones, or a nucleic acid molecule having a sequencecomplementary to one of the above sequences. Such isolated molecules,particularly DNA molecules, are useful, for example, as probes for genemapping by in situ hybridization with chromosomes, and for detectingexpression of the TNFR genes in human tissue, for instance, by Northernblot analysis.

The present invention is further directed to nucleic acid moleculesencoding portions of the nucleotide sequences described herein as wellas to fragments of the isolated nucleic acid molecules described herein.In particular, the invention provides polynucleotides having anucleotide sequence representing the portion of SEQ ID NO:1 or 3 whichconsist of positions 25-924 and 73-582 of SEQ ID NOS:1 and 3,respectively. Also contemplated are polynucleotides encoding TNFRpolypeptides which lack an amino terminal methionine suchpolynucleotides having a nucleotide sequence representing the portion ofSEQ ID NOS:1 and 3 which consist of positions 28-924 and 76-582,respectively. Polypeptides encoded by such polynucleotides are alsoprovided, such polypeptides comprising an amino acid sequence atpositions 2-300 and 2-170 of SEQ ID NOS:2 and 4, respectively.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NOS:1 and 3as follows: HELDI06R (SEQ ID NO:17) and HCEOW38R (SEQ ID NO:18) arerelated to both SEQ ID NOS:1 and 3. Preferred are polynucleotidefragments of SEQ ID NOS:1 and 3 which are not SEQ ID NO:17 or 18 orsubfragments of either SEQ ID NO:17 or 18. The sequences of HELDI06R andHCEOW38R are shown in FIG. 6.

More generally, by a fragment of an isolated nucleic acid moleculehaving the nucleotide sequence of the deposited cDNA or the nucleotidesequence shown in FIG. 1 or 2 (SEQ ID NOS:1 or 3) is intended fragmentsat least about 15 nt, and more preferably at least about 20 nt, stillmore preferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length. These fragments have numerous uses, whichinclude, but are not limited to, as diagnostic probes and primers asdiscussed herein. Of course, larger fragments 50-300 nt in length arealso useful according to the present invention as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNAs or as shown in FIGS. 1 and 2A-B (SEQ ID NOS:1 and 3).Especially preferred are fragments comprising at least 500 nucleotideswhich are at least 80%, 85%, 90%, 92%, or 95% identical to 500contiguous nucleotides shown in SEQ ID NO:1. By a fragment at leastabout 20 nt in length, for example, is intended fragments which include20 or more contiguous bases from the nucleotide sequence of a depositedcDNA or the nucleotide sequence as shown in FIGS. 1 and 2A-B (SEQ IDNOS:1 and 3). In this context “about” includes the particularly recitedsize, and those sizes that are larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini. Preferrednucleic acid fragments of the present invention include nucleic acidmolecules encoding epitope-bearing portions of the TNFR polypeptides asidentified in FIGS. 4 and 5 and described in more detail below.

Representative examples of TNFR-6α nucleic acid fragments of theinvention include, for example,fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to aboutnucleotide 25, about nucleotide 26 to about nucleotide 75, aboutnucleotide 76 to about nucleotide 114, about nucleotide 115 to aboutnucleotide 162, about nucleotide 163 to about nucleotide 216, aboutnucleotide 217 to about nucleotide 267, about nucleotide 268 to aboutnucleotide 318, about nucleotide 319 to about nucleotide 369, aboutnucleotide 370 to about nucleotide 420, about nucleotide 421 to aboutnucleotide 471, about nucleotide 472 to about nucleotide 522, aboutnucleotide 523 to about nucleotide 573, about nucleotide 574 to aboutnucleotide 625, about nucleotide 626 to about nucleotide 675, aboutnucleotide 676 to about nucleotide 714, about nucleotide 715 to aboutnucleotide 765, about nucleotide 766 to about nucleotide 816, aboutnucleotide 817 to about nucleotide 867, about nucleotide 868 to aboutnucleotide 924, about nucleotide 925 to about nucleotide 975 of SEQ IDNO:1, or the complementary strand thereto, or the cDNA contained in theplasmid deposited as ATCC Deposit No. 97810. In this context “about”includes the particularly recited ranges, and those ranges that arelarger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini.

In specific embodiments, the nucleic acid fragments of the inventioncomprise, or alternatively, consist of, a polynucleotide sequenceencoding amino acid residues 100 to 150, 150 to 200, 200 to 300, 220 to300, 240 to 300, 250 to 300, 260 to 300, and/or 280 to 300, of SEQ IDNO:2, or the complementary strand thereto. Polynucleotides thathybridize to these polynucleotide fragments are also encompassed by theinvention.

Representative examples of TNFR—6β nucleic acid fragments of theinvention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to aboutnucleotide 36, about nucleotide 37 to about nucleotide 72, aboutnucleotide 73 to about nucleotide 123, about nucleotide 124 to aboutnucleotide 175, about nucleotide 176 to about nucleotide 216, aboutnucleotide 217 to about nucleotide 267, about nucleotide 268 to aboutnucleotide 318, about nucleotide 319 to about nucleotide 369, aboutnucleotide 370 to about nucleotide 420, about nucleotide 421 to aboutnucleotide 471, about nucleotide 472 to about nucleotide 522, aboutnucleotide 523 to about nucleotide 582, about nucleotide 583 to aboutnucleotide 622, about nucleotide 623 to about nucleotide 682, aboutnucleotide 683 to about nucleotide 750, about nucleotide 751 to aboutnucleotide 800, about nucleotide 801 to about nucleotide 850, aboutnucleotide 851 to about nucleotide 900, about nucleotide 901 to aboutnucleotide 950, about nucleotide 951 to about nucleotide 1000, aboutnucleotide 1001 to about nucleotide 1050, about nucleotide 1051 to aboutnucleotide 1100, about nucleotide 1101 to about nucleotide 1150, aboutnucleotide 1151 to about nucleotide 1200, about nucleotide 1201 to aboutnucleotide 1250, about nucleotide 1251 to about nucleotide 1300, aboutnucleotide 1301 to about nucleotide 1350, about nucleotide 1351 to aboutnucleotide 1400, about nucleotide 1401 to about nucleotide 1450, aboutnucleotide 1451 to about nucleotide 1500, about nucleotide 1501 to aboutnucleotide 1550, about nucleotide 1551 to about nucleotide 1600 aboutnucleotide 1601 to about nucleotide 1650, about nucleotide 1651 to aboutnucleotide 1667 of SEQ ID NO:3, or the complementary strand thereto, orthe cDNA contained in the plasmid deposited as ATCC Deposit No. 97809.In this context “about” includes the particularly recited ranges, andthose ranges that are larger or smaller by several (5, 4, 3, 2, or 1)nucleotides, at either terminus or at both termini.

In specific embodiments, the nucleic acid fragments of the inventioncomprise, or alternatively, consist of, a polynucleotide sequenceencoding amino acid residues 50 to 100, 100 to 170, 110 to 170, 130 to170, 140 to 170, 150 to 170, and/or 160 to 170, of SEQ ID NO:4, or thecomplementary strand thereto. Polynucleotides that hybridize to thesepolynucleotide fragments are also encompassed by the invention.

Preferably, the polynucleotide fragments of the invention encode apolypeptide which demonstrates a TNFR-6α and/or TNFR-6β functionalactivity. By a polypeptide demonstrating “functional activity” is meant,a polypeptide capable of displaying one or more known functionalactivities associated with a complete (full-length) or mature TNFR-6αand/or TNFR-6β polypeptide. Such functional activities include, but arenot limited to, biological activity (e.g., inhibition or reduction ofFasL mediated apoptosis, inhibition or reduction of AIM-II mediatedapoptosis), antigenicity [ability to bind (or compete with a TNFR-6αand/or TNFR-6β polypeptide for binding) to an anti-TNFR-6α antibodyand/or anti-TNFR-6β antibody], immunogenicity (ability to generateantibody which binds to a TNFR-6α and/or TNFR-6β polypeptide), abilityto form multimers with TNFR-6α and/or TNFR-6β polypeptides of theinvention, and ability to bind to a receptor or ligand for a TNFR-6αand/or TNFR-6β polypeptide (e.g., Fas ligand and/or AIM-II(International application publication number WO 97/34911, publishedSep. 25, 1997)).

The functional activity of TNFR-6α and/or TNFR-6β polypeptides, andfragments, variants derivatives, and analogs thereof, can be assayed byvarious methods.

For example, in one embodiment where one is assaying for the ability tobind or compete with complete (full-length) or mature TNFR-6α and/orTNFR-6β polypeptide for binding to anti-TNFR-6α and/or anti-TNFR-6βantibody, various immunoassays known in the art can be used, includingbut not limited to, competitive and non-competitive assay systems usingtechniques such as radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitation reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labelled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

In another embodiment, where a TNF-ligand is identified (e.g., FasLigand and/or AIM-II (International application publication number WO97/34911, published Sep. 25, 1997)), or the ability of a polypeptidefragment, variant or derivative of the invention to multimerize is beingevaluated, binding can be assayed, e.g., by means well-known in the art,such as, for example, reducing and non-reducing gel chromatography,protein affinity chromatography, and affinity blotting. See generally,Phizicky, E., et al., Microbiol. Rev. 59:94-123 (1995). In anotherembodiment, physiological correlates of TNFR-6α and/or TNFR-6β bindingto its substrates (signal transduction) can be assayed.

In addition, assays described herein (e.g., see Examples 7-9) andotherwise known in the art may routinely be applied or modified tomeasure the ability of TNFR-6α and/or TNFR-6β polypeptides andfragments, variants derivatives and analogs thereof, to elicit TNFR-6αand/or TNFR-6β related biological activity (e.g., to inhibit or reduceFasL mediated apoptosis in vitro or in vivo, or to inhibit or reduceAIM-II mediated apoptosis in vitro or in vivo).

For example, the ability of TNFR polypeptides of the invention to reduceor block FasL mediated apoptosis can be assayed using a Fas expressingT-cell line, such as Jurkat. In this assay, Jurkat cells treated withsoluble FasL undergo apoptosis. Pretreatment of cells with TNFR and/orTNFR agonists prior to addition of FasL protects cells from undergoingapoptosis and results in a reduced level of apoptosis when compared tothat observed when the same concentration of soluble FasL is contactedwith the same concentration of the Fas expressing cells in the absenceof the TNFR polypeptide or TNFR agonist. Alternatively mixing of theFasL protein with TNFR and/or TNFR agonist will also block the abilityof FasL to bind the Jurkat cells and mediate apoptosis (see, e.g.,Example 9).

In contrast, TNFR antagonists of the invention block TNFR mediatedinhibition of FasL mediated apoptosis. Accordingly, TNFR antagonists ofthe invention can be assayed, for example, by combining the mature TNFR(known to bind FasL), the TNFR antagonist to be tested, and solubleFasL, and contacting this combination with the Fas expressing cell line.TNFR antagonists reduce or block TNFR mediated inhibition of FasLmediated apoptosis. Accordingly, Fas expressing T cells contacted withmature TNFR, TNFR antagonist and soluble FasL exhibit elevated apoptosislevels when compared with the same concentration of Fas expressing cellsthat have been contacted with the same concentrations of mature TNFR andFasL in the absence of the TNFR antagonist.

Apoptosis can be measured, for example, by increased staining withAnnexin, which selectively binds apoptotic cells. In another example,the decrease in cell numbers due to apoptosis can be detected by adecrease in ALOMAR blue staining which detects viable cells.

Other methods will be known to the skilled artisan and are within thescope of the invention.

In additional embodiments, the polynucleotides of the invention encodefunctional attributes of TNFR-6α and/or TNFR-6β. Preferred embodimentsof the invention in this regard include fragments that comprisealpha-helix and alpha-helix forming regions (“alpha-regions”),beta-sheet and beta-sheet forming regions (“beta-regions”), turn andturn-forming regions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions and high antigenic index regions of TNFR-6αand/or TNFR-6β polypeptides.

Certain preferred regions in this regard are set out in FIG. 4 (Table I)and FIG. 5 (Table II). The data presented in FIG. 4 and FIG. 5 and thatpresented in Table I and Table II, respectively, merely present adifferent format of the same results obtained when the amino acidsequence of SEQ ID NO:2 and the amino acid sequence of SEQ ID NO:4 isanalyzed using the default parameters of the DNA*STAR computeralgorithm.

The above-mentioned preferred regions set out in FIG. 4 (Table I) andFIG. 5 (Table II) include, but are not limited to, regions of theaforementioned types identified by analysis of the amino acid sequenceset out in FIG. 1 and FIG. 2A. As set out in FIG. 4 (Table I) and FIG. 5(Table II), such preferred regions include Garnier-Robson alpha-regions,beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions,beta-regions, and coil-regions, Kyte-Doolittle hydrophilic regions,Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexibleregions, Emini surface-forming regions and Jameson-Wolf regions of highantigenic index. Among highly preferred polynucleotides in this regardare those that encode polypeptides comprising regions of TNFR-6α and/orTNFR-6β that combine several structural features, such as several (e.g.,1, 2, 3, or 4) of the features set out above.

Additionally, the data presented in columns VIII, IX, XIII, and XIV ofTables I and II can routinely be used to determine regions of TNFR-6αand/or TNFR-6β which exhibit a high degree of potential forantigenicity. Regions of high antigenicity are determined from the datapresented in columns VIII, IX, XIII, and/or XIV by choosing values whichrepresent regions of the polypeptide which are likely to be exposed onthe surface of the polypeptide in an environment in which antigenrecognition may occur in the process of initiation of an immuneresponse.

TABLE I Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met1 . . B . . . . 0.06 0.09 * . . −0.10 0.60 Arg 2 . . B . . . . 0.10−0.34 * . . 0.50 0.82 Ala 3 . . B . . . . 0.28 −0.34 * . . 0.50 0.63 Leu4 A . . . . . . 0.32 −0.34 * . . 0.50 0.99 Glu 5 A . . . . . . −0.10−0.53 * . F 0.95 0.50 Gly 6 . . . . . T C 0.20 0.16 * . F 0.45 0.41 Pro7 . . . . T T . −0.72 0.04 * . F 0.65 0.66 Gly 8 . . . . T T . −0.940.04 . . F 0.65 0.32 Leu 9 A . . . . T . −0.80 0.73 . . . −0.20 0.26 Ser10 A A . . . . . −1.61 0.87 . . . −0.60 0.09 Leu 11 . A B . . . . −2.121.13 . . . −0.60 0.08 Leu 12 . A B . . . . −2.72 1.34 . . . −0.60 0.07Cys 13 . A B . . . . −2.97 1.34 . . . −0.60 0.04 Leu 14 . A B . . . .−2.97 1.46 . . . −0.60 0.05 Val 15 . A B . . . . −2.88 1.46 . . . −0.600.05 Leu 16 . A B . . . . −2.66 1.20 . . . −0.60 0.15 Ala 17 . A B . . .. −2.66 1.13 . . . −0.60 0.18 Leu 18 . A B . . . . −2.80 1.13 . . .−0.60 0.20 Pro 19 A A . . . . . −2.20 1.17 . . . −0.60 0.20 Ala 20 . A B. . . . −2.20 0.91 . . . −0.60 0.31 Leu 21 . A B . . . . −1.60 1.06 . *. −0.60 0.28 Leu 22 . A B . . . . −1.60 0.80 . . . −0.60 0.28 Pro 23 . AB . . . . −1.64 0.87 . * . −0.60 0.28 Val 24 . . B . . . . −1.32 1.01. * . −0.40 0.25 Pro 25 . . B . . . . −1.08 0.33 * . . −0.10 0.60 Ala 26. . B B . . . −1.12 0.07 . . . −0.30 0.38 Val 27 . . B B . . . −0.900.29 * * . −0.30 0.38 Arg 28 . . B B . . . −0.69 0.14 * * . −0.30 0.25Gly 29 . . B B . . . −0.14 −0.29 * * . 0.30 0.43 Val 30 . . B B . . .−0.14 −0.30 * * . 0.30 0.83 Ala 31 . . B B . . . 0.13 −0.51 * * . 0.600.66 Glu 32 . . B . . . . 0.74 −0.03 * * F 0.65 0.96 Thr 33 . . B . . T. 0.42 0.30 * * F 0.40 2.02 Pro 34 . . . . T T . 0.48 0.09 * * F 0.803.10 Thr 35 . . . . T T . 1.44 0.50 * . F 0.50 1.88 Tyr 36 . . . . . T C2.03 0.50 * . . 0.15 2.55 Pro 37 . . . . T . . 1.44 0.01 * . . 0.45 2.76Trp 38 . A . . . . C 1.76 0.09 * . . 0.05 1.93 Arg 39 . A B . . . . 1.66−0.40 * . F 0.60 2.13 Asp 40 A A . . . . . 1.62 −0.67 * . F 0.90 1.99Ala 41 . A . . . . C 1.87 −0.67 * . F 1.10 1.87 Glu 42 A A . . . . .2.19 −1.59 * * F 0.90 1.66 Thr 43 A A . . . . . 1.67 −1.59 * . F 0.901.94 Gly 44 . A . . T . . 0.70 −0.90 * . F 1.30 1.59 Glu 45 A A . . . .. 0.03 −0.76 * * F 0.75 0.68 Arg 46 A A . . . . . 0.03 −0.19 * . F 0.450.25 Leu 47 A A . . . . . 0.03 −0.17 * . . 0.30 0.26 Val 48 . A B . . .. −0.32 −0.20 . . . 0.30 0.26 Cys 49 . A B . . . . −0.19 0.37 . . .−0.30 0.07 Ala 50 . A B . . . . −0.40 0.80 . . . −0.60 0.13 Gln 51 . A B. . . . −0.86 0.54 . . . −0.60 0.28 Cys 52 . A B . . . . −0.36 0.33 . .. −0.30 0.51 Pro 53 . . . . . T C −0.20 0.24 . . F 0.45 0.73 Pro 54 . .. . T T . −0.39 0.53 . . F 0.35 0.36 Gly 55 . . . . T T . 0.20 0.77 * .F 0.35 0.50 Thr 56 . . B . . T . 0.31 0.60 . . F −0.05 0.56 Phe 57 . . BB . . . 0.77 0.17 * . F −0.15 0.71 Val 58 . . B B . . . 0.31 0.17 * . .0.19 1.12 Gln 59 . . B B . . . 0.63 0.31 * . F 0.53 0.41 Arg 60 . . B .. T . 1.09 −0.17 * . F 1.87 0.94 Pro 61 . . B . . T . 1.40 −0.96 * . F2.66 2.47 Cys 62 . . . . T T . 1.80 −1.60 * . F 3.40 2.38 Arg 63 . . . .T T . 2.44 −1.61 * . F 3.06 1.63 Arg 64 . . . . T . . 2.13 −1.19 * . F2.77 1.63 Asp 65 . . . . T . . 1.71 −1.13 * . F 2.68 4.39 Ser 66 . . . .T T . 1.26 −1.21 . . F 2.79 3.24 Pro 67 . . . . T T . 1.58 −0.64 . . F2.55 0.89 Thr 68 . . . . T T . 1.26 −0.21 . . F 2.50 0.52 Thr 69 . . . .T T . 0.48 0.21 . . F 1.65 0.61 Cys 70 . . . . T . . 0.27 0.40 . . F1.14 0.21 Gly 71 . . . . T T . 0.36 0.40 * * F 1.33 0.22 Pro 72 . . . .T T . 0.68 0.34 * . F 1.62 0.24 Cys 73 . . . . . T C 0.96 −0.14 * . F2.01 0.88 Pro 74 . . . . . T C 1.02 −0.21 * . F 2.40 1.21 Pro 75 . . . .T T . 1.38 0.11 * * F 1.76 1.23 Arg 76 . . . . T T . 1.72 0.17 * * F1.52 3.30 His 77 . . B . . T . 1.23 0.00 * * F 0.88 3.70 Tyr 78 . . B .. T . 1.61 0.36 * * . 0.49 2.07 Thr 79 . . B . . . . 1.82 0.84 * . .−0.25 1.11 Gln 80 . . B . . . . 1.79 1.24 * * . −0.25 1.31 Phe 81 . . .. T . . 0.87 1.50 * * . 0.15 1.31 Trp 82 . . . . T . . 0.90 1.43 * . .0.00 0.75 Asn 83 . A . . T . . 1.26 0.94 * . . −0.20 0.75 Tyr 84 . A . .T . . 0.90 0.54 * * . −0.05 1.70 Leu 85 . A . . T . . 1.01 0.33 * * .0.38 0.87 Glu 86 . A . . T . . 1.47 −0.59 * * . 1.71 1.05 Arg 87 . A . .T . . 1.09 −0.23 . * . 1.69 1.05 Cys 88 . . . . T T . 1.09 −0.41 . * .2.22 0.69 Arg 89 . . . . T T . 0.48 −0.70 . * . 2.80 0.64 Tyr 90 . . . .T T . 0.48 −0.06 . * . 2.22 0.24 Cys 91 . . . . T T . −0.19 0.63 . * .1.04 0.37 Asn 92 . . B B . . . −0.64 0.63 . * . −0.04 0.10 Val 93 . . BB . . . 0.02 1.06 . * . −0.32 0.06 Leu 94 . . B B . . . 0.02 0.30 . . .−0.30 0.21 Cys 95 . . B . . T . 0.27 −0.27 . . . 0.70 0.25 Gly 96 . . .. . T C 0.93 −0.67 . . F 1.35 0.59 Glu 97 A . . . . T . 0.93 −1.31 . . F1.30 1.24 Arg 98 A . . . . T . 1.20 −2.00 . * F 1.30 4.00 Glu 99 A A . .. . . 2.12 −2.07 . * F 0.90 4.08 Glu 100 A A . . . . . 2.20 −2.50 . * F0.90 4.61 Glu 101 A A . . . . . 1.88 −2.00 . * F 0.90 2.38 Ala 102 A A .. . . . 1.84 −1.43 . . F 0.75 0.74 Arg 103 A A . . . . . 1.14 −0.93 . .. 0.60 0.58 Ala 104 A A . . . . . 0.83 −0.43 . * . 0.30 0.34 Cys 105 A A. . . . . 0.80 0.06 . * . −0.30 0.48 His 106 A A . . . . . 0.80 0.06 * *. −0.30 0.34 Ala 107 A A . . . . . 1.50 0.46 * * . −0.60 0.53 Thr 108 AA . . . . . 0.80 −0.04 * * . 0.45 1.95 His 109 . A . . T . . 0.72−0.11 * . . 1.13 1.45 Asn 110 . A . . T . . 1.50 −0.04 * . . 1.26 0.77Arg 111 . A . . T . . 0.87 −0.54 . * . 1.99 1.04 Ala 112 . A . . T . .1.57 −0.46 . * . 1.82 0.41 Cys 113 . . . . T T . 1.57 −0.96 . * . 2.800.50 Arg 114 . . B . . T . 1.26 −0.87 * * . 2.12 0.37 Cys 115 . . . . TT . 0.56 −0.44 * * . 1.94 0.36 Arg 116 . . . . T T . −0.26 −0.16 . * .1.66 0.58 Thr 117 . A . B T . . −0.26 0.06 . * F 0.53 0.26 Gly 118 . A .B T . . 0.38 0.56 . * . −0.20 0.49 Phe 119 . A B B . . . −0.32 0.49 . *. −0.60 0.34 Phe 120 . A B B . . . −0.00 0.99 . * . −0.60 0.24 Ala 121 AA . B . . . −0.81 0.93 . * . −0.60 0.24 His 122 A A . . . . . −1.17 1.29. . . −0.60 0.24 Ala 123 A A . . . . . −1.63 1.07 . * . −0.60 0.15 Gly124 A A . . . . . −0.93 0.97 . * . −0.60 0.12 Phe 125 A A . . . . .−0.27 0.47 . . . −0.60 0.15 Cys 126 A A . . . . . −0.27 0.47 . * . −0.600.20 Leu 127 A A . . . . . −0.53 0.47 . . . −0.60 0.21 Glu 128 A A . . .. . −0.61 0.43 . . . −0.60 0.32 His 129 . . . . T T . −0.48 0.21 . . .0.50 0.32 Ala 130 . . . . T T . 0.01 0.07 . . . 0.63 0.61 Ser 131 . . .. T T . 0.33 −0.19 . . . 1.36 0.54 Cys 132 . . . . . T C 0.56 0.24 . . .0.69 0.39 Pro 133 . . . . . T C 0.21 0.24 . . F 0.97 0.39 Pro 134 . . .. T T . −0.61 0.17 . . F 1.30 0.29 Gly 135 . . . . T T . −0.91 0.43 . .F 0.87 0.40 Ala 136 . . B . . T . −1.20 0.54 . . . 0.19 0.18 Gly 137 . .B B . . . −0.74 0.61 . . . −0.34 0.12 Val 138 . . B B . . . −0.88 0.61 .. . −0.47 0.19 Ile 139 . . B B . . . −0.98 0.61 . . . −0.60 0.18 Ala 140. . B B . . . −0.84 0.60 . . . −0.60 0.27 Pro 141 . . B . . . . −0.560.60 . . F −0.25 0.55 Gly 142 . . . . T . . −0.21 0.34 . . F 0.88 1.06Thr 143 . . . . . T C 0.64 0.06 . . F 1.16 1.82 Pro 144 . . . . . T C1.22 −0.04 . . F 2.04 1.89 Ser 145 . . . . T T . 1.81 0.01 . . F 1.922.76 Gln 146 . . . . T T . 1.36 −0.01 . . F 2.80 3.31 Asn 147 . . . . TT . 1.70 0.07 . . F 1.92 1.15 Thr 148 . . . . T T . 1.80 0.04 . . F 1.641.48 Gln 149 . . . . T T . 1.34 0.09 . . F 1.36 1.32 Cys 150 . . B . . T. 1.43 0.26 . . F 0.53 0.44 Gln 151 . . B . . . . 1.22 0.29 . . F 0.050.47 Pro 152 . . B . . . . 0.88 0.23 . * F 0.05 0.42 Cys 153 . . B . . .. 0.88 0.26 . * F 0.05 0.78 Pro 154 . . B . . T . 0.18 0.17 . * F 0.250.65 Pro 155 . . . . T T . 0.54 0.56 . * F 0.35 0.36 Gly 156 . . . . T T. −0.04 0.51 . * F 0.35 0.91 Thr 157 . . B . . T . −0.13 0.44 . . F−0.05 0.59 Phe 158 . . B . . . . 0.23 0.40 . . F −0.25 0.51 Ser 159 . .B . . . . 0.14 0.36 . . F 0.39 0.70 Ala 160 . . B . . . . 0.06 0.31 . .F 0.73 0.65 Ser 161 . . . . . T C 0.10 0.21 . . F 1.62 1.00 Ser 162 . .. . . T C 0.41 −0.19 . . F 2.56 1.00 Ser 163 . . . . T T . 1.11 −0.57 .. F 3.40 1.72 Ser 164 . . . . T T . 0.74 −0.67 . . F 3.06 2.22 Ser 165 .. . . T . . 1.33 −0.49 . . F 2.07 0.89 Glu 166 . . . . T . . 1.42 −0.47. . F 1.88 1.15 Gln 167 . . . . T . . 1.69 −0.43 . . F 1.82 1.32 Cys 168. . . . T . . 2.10 −0.31 . . F 1.76 1.34 Gln 169 . . B . . . . 2.40−0.70 . . F 1.94 1.52 Pro 170 . . . . T . . 2.03 −0.30 . . F 2.32 1.41His 171 . . . . T T . 1.72 −0.13 . . F 2.80 1.41 Arg 172 . . . . T T .1.13 −0.21 . . F 2.52 1.18 Asn 173 . . . . T T . 0.99 −0.11 * . . 1.940.77 Cys 174 . . B . . T . 0.64 0.14 . . . 0.66 0.47 Thr 175 . A B . . .. 0.04 0.07 . . . −0.02 0.24 Ala 176 . A B . . . . −0.51 0.76 * . .−0.60 0.12 Leu 177 . A B . . . . −1.43 0.86 * . . −0.60 0.23 Gly 178 . AB . . . . −1.43 0.97 . * . −0.60 0.13 Leu 179 . A B . . . . −1.62 0.89. * . −0.60 0.21 Ala 180 . A B . . . . −1.52 1.03 . * . −0.60 0.19 Leu181 . A B . . . . −1.28 0.77 . * . −0.60 0.29 Asn 182 . A B . . . .−0.77 0.77 . * . −0.60 0.35 Val 183 . . B . . T . −0.72 0.47 . * F −0.050.46 Pro 184 . . . . . T C −0.21 0.36 . * F 0.73 0.75 Gly 185 . . . . TT . 0.34 0.06 . * F 1.21 0.63 Ser 186 . . . . T T . 1.16 0.16 . * F 1.641.15 Ser 187 . . . . . T C 0.84 −0.49 . . F 2.32 1.24 Ser 188 . . . . TT . 0.89 −0.43 . . F 2.80 1.81 His 189 . . B . . T . 0.43 −0.17 . . F2.12 1.11 Asp 190 . . . . T T . 0.47 0.01 . . F 1.49 0.45 Thr 191 . . B. . . . 0.47 0.11 . . F 0.61 0.48 Leu 192 . . B . . . . 0.10 0.11 . . .0.18 0.47 Cys 193 . . B . . T . 0.09 0.19 . . . 0.10 0.15 Thr 194 . . B. . T . −0.22 0.67 . . . −0.20 0.15 Ser 195 . . B . . T . −0.92 0.61 * .F −0.05 0.18 Cys 196 . . B . . T . −0.82 0.71 . . F −0.05 0.29 Thr 197 .. . . T . . −0.82 0.57 . . F 0.15 0.31 Gly 198 . . . . T . . −0.46 0.77. . . 0.00 0.19 Phe 199 . . B . . . . −0.46 0.77 . * . −0.40 0.48 Pro200 . . B . . . . −0.04 0.69 * * . −0.40 0.48 Leu 201 . . B . . . .−0.23 0.20 * * . −0.10 0.96 Ser 202 . . B . . . . −0.13 0.41 * * F 0.020.82 Thr 203 . . B . . . . −0.13 0.06 . * F 0.59 0.82 Arg 204 . . . . .. C −0.02 0.06 . * F 1.06 0.99 Val 205 . . . . . T C 0.19 −0.13 . * F2.13 0.74 Pro 206 . . . . . T C 1.00 −0.51 . * F 2.70 0.89 Gly 207 . . .. . T C 0.63 −1.00 . * F 2.43 0.79 Ala 208 A . . . . T . 0.94 −0.43 . *F 1.66 0.57 Glu 209 A A . . . . . 0.94 −1.07 . * F 1.29 0.64 Glu 210 A A. . . . . 1.21 −1.50 * . F 1.17 1.26 Cys 211 A A . . . . . 0.57 −1.43 *. F 0.90 1.26 Glu 212 A A . . . . . 0.02 −1.29 * * F 0.75 0.54 Arg 213 AA . . . . . 0.61 −0.60 * * . 0.60 0.22 Ala 214 A A . . . . . −0.09−0.60 * * . 0.60 0.68 Val 215 A A . . . . . −0.94 −0.39 * * . 0.30 0.34Ile 216 A A . . . . . −0.87 0.26 * * . −0.30 0.13 Asp 217 A A . . . . .−1.57 0.76 * * . −0.60 0.13 Phe 218 A A . . . . . −1.68 1.04 * * . −0.600.15 Val 219 A A . . . . . −1.09 0.80 . . . −0.60 0.37 Ala 220 A A . . .. . −1.12 0.11 . . . −0.30 0.37 Phe 221 A A . . . . . −0.53 0.80 . * .−0.60 0.30 Gln 222 A A . . . . . −1.42 0.40 . * . −0.60 0.54 Asp 223 A A. . . . . −0.68 0.44 . . F −0.45 0.38 Ile 224 A A . . . . . 0.29 −0.06 .. F 0.45 0.87 Ser 225 A A . . . . . 0.07 −0.84 . . F 0.75 0.99 Ile 226 AA . . . . . 0.77 −0.56 * . F 0.75 0.49 Lys 227 A A . . . . . 0.88−0.16 * * F 0.60 1.20 Arg 228 A A . . . . . 0.07 −0.84 * * F 0.90 1.76Leu 229 A A . . . . . 0.14 −0.54 * . F 0.90 2.07 Gln 230 A A . . . . .0.44 −0.54 * . F 0.75 0.85 Arg 231 . A B . . . . 0.74 −0.14 * . . 0.300.76 Leu 232 A A . . . . . −0.11 0.36 * . . −0.30 0.93 Leu 233 . A B . .. . −0.22 0.36 * * . −0.30 0.44 Gln 234 . A B . . . . −0.00 −0.04 * . .0.30 0.39 Ala 235 . A B . . . . −0.21 0.46 * . . −0.60 0.48 Leu 236 . AB . . . . −0.32 0.20 * * . −0.30 0.89 Glu 237 . A B . . . . 0.14 −0.49 .. . 0.30 0.89 Ala 238 . . B . . T . 0.67 −0.46 . . F 0.85 0.88 Pro 239 .. . . T T . 0.32 −0.04 . . F 1.40 1.12 Glu 240 . . . . T T . 0.70 −0.30. . F 1.25 0.64 Gly 241 . . . . T T . 1.20 0.13 . . F 0.65 0.98 Trp 242. . . . T . . 0.99 0.11 * . F 0.45 0.91 Gly 243 . . . . . . C 1.690.11 * * F 0.59 0.81 Pro 244 . . . . . . C 1.31 0.11 * * F 1.08 1.61 Thr245 . . . . . T C 0.97 0.19 * . F 1.62 1.55 Pro 246 . . . . . T C 1.42−0.30 * . F 2.56 1.55 Arg 247 . . . . T T . 1.12 −0.73 * . F 3.40 1.96Ala 248 . . . . . T C 0.88 −0.66 * . F 2.86 1.37 Gly 249 A A . . . . .0.28 −0.64 * * F 1.77 0.90 Arg 250 A A . . . . . 0.59 −0.39 * * . 0.980.38 Ala 251 A A . . . . . −0.01 0.01 * * . 0.04 0.65 Ala 252 A A . . .. . −0.08 0.20 * * . −0.30 0.54 Leu 253 A A . . . . . −0.30 −0.23 * * .0.30 0.55 Gln 254 A A . . . . . 0.16 0.46 . * . −0.60 0.45 Leu 255 A A .. . . . 0.16 −0.04 . * . 0.30 0.87 Lys 256 A A . . . . . 0.86 −0.54 . *. 0.75 2.07 Leu 257 A A . . . . . 0.63 −1.23 . * F 0.90 2.34 Arg 258 A A. . . . . 1.13 −0.94 * * F 0.90 2.34 Arg 259 . A B . . . . 1.13−1.14 * * F 0.90 1.69 Arg 260 . A B . . . . 1.13 −1.14 * * F 0.90 3.55Leu 261 . A B . . . . 0.28 −1.14 * * F 0.90 1.49 Thr 262 . A B . . . .0.74 −0.46 * * F 0.45 0.63 Glu 263 . A B . . . . 0.04 −0.03 * * . 0.300.32 Leu 264 . A B . . . . −0.07 0.47 * . . −0.60 0.39 Leu 265 . A B . .. . −0.18 0.19 . * . −0.30 0.47 Gly 266 A A . . . . . 0.29 −0.30 . . .0.30 0.45 Ala 267 A . . . . T . 0.01 0.13 . . F 0.25 0.54 Gln 268 A . .. . T . −0.80 −0.06 . . F 0.85 0.66 Asp 269 A . . . . T . −0.80 −0.06 .. F 0.85 0.55 Gly 270 A . . . . T . −0.84 0.20 * * . 0.10 0.45 Ala 271 AA . . . . . −0.39 0.34 * * . −0.30 0.19 Leu 272 . A B . . . . −0.61−0.06 * * . 0.30 0.23 Leu 273 . A B . . . . −1.42 0.63 * * . −0.60 0.19Val 274 A A . . . . . −1.42 0.89 * * . −0.60 0.15 Arg 275 A A . . . . .−1.67 0.79 * * . −0.60 0.32 Leu 276 A A . . . . . −1.89 0.60 * * . −0.600.40 Leu 277 A A . . . . . −0.97 0.60 * * . −0.60 0.44 Gln 278 A A . . .. . −1.01 −0.04 * * . 0.30 0.44 Ala 279 A A . . . . . −0.74 0.60 * * .−0.60 0.40 Leu 280 A A . . . . . −0.74 0.41 * * . −0.60 0.49 Arg 281 . AB . . . . −0.53 −0.27 * . . 0.30 0.55 Val 282 . A B . . . . 0.07 −0.06 *. . 0.30 0.54 Ala 283 . A B . . . . −0.28 −0.13 * . . 0.72 1.01 Arg 284. A B . . . . −0.50 −0.39 * . . 0.84 0.51 Met 285 . . B . . T . 0.310.30 . * . 0.91 0.57 Pro 286 . . . . . T C 0.31 −0.34 . * F 2.13 0.97Gly 287 . . . . . T C 0.87 −0.84 * * F 2.70 0.97 Leu 288 A . . . . T .0.60 −0.46 * * F 2.08 1.32 Glu 289 A . . . . . . 0.60 −0.43 * * F 1.460.63 Arg 290 A . . . . . . 1.20 −0.86 * * F 1.64 1.25 Ser 291 A . . . .. . 1.52 −1.29 * * F 1.37 2.62 Val 292 A . . . . . . 1.17 −1.97 * * F1.10 2.97 Arg 293 A . . . . . . 1.17 −1.19 * * F 1.10 1.31 Glu 294 A . .. . . . 0.96 −0.50 * * F 0.65 0.81 Arg 295 A . . . . . . −0.01 −0.46 * *F 0.80 1.68 Phe 296 . . B . . . . 0.26 −0.46 . * . 0.50 0.64 Leu 297 . .B . . . . 0.72 0.04 . * . −0.10 0.50 Pro 298 A . . . . . . 0.22 0.47 . *. −0.40 0.33 Val 299 A . . . . . . −0.17 0.90 * . . −0.40 0.48 His 300 A. . . . . . −0.67 0.54 . . . −0.40 0.75

TABLE II Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met1 . . B . . . . 0.06 0.09 * . . −0.10 0.60 Arg 2 . . B . . . . 0.10−0.34 * . . 0.50 0.82 Ala 3 . . B . . . . 0.28 −0.34 * . . 0.50 0.63 Leu4 . . B . . . . 0.32 −0.34 . . . 0.50 0.99 Glu 5 . . B . . . . −0.10−0.53 . . F 0.95 0.50 Gly 6 . . . . . T C 0.20 0.16 * . F 0.45 0.41 Pro7 . . . . T T . −0.72 0.04 * . F 0.65 0.66 Gly 8 . . . . T T . −0.940.04 . . F 0.65 0.32 Leu 9 . . B . . T . −0.80 0.73 . . . −0.20 0.26 Ser10 . A B . . . . −1.61 0.87 . . . −0.60 0.09 Leu 11 . A B . . . . −2.121.13 . . . −0.60 0.08 Leu 12 . A B . . . . −2.72 1.34 . . . −0.60 0.07Cys 13 . A B . . . . −2.97 1.34 . . . −0.60 0.04 Leu 14 . A B . . . .−2.97 1.46 . . . −0.60 0.05 Val 15 . A B . . . . −2.88 1.46 . . . −0.600.05 Leu 16 . A B . . . . −2.66 1.20 . . . −0.60 0.15 Ala 17 . A B . . .. −2.66 1.13 . . . −0.60 0.18 Leu 18 . A B . . . . −2.80 1.13 . . .−0.60 0.20 Pro 19 . A B . . . . −2.20 1.17 . . . −0.60 0.20 Ala 20 . A B. . . . −2.20 0.91 . . . −0.60 0.31 Leu 21 . A B . . . . −1.60 1.06 . *. −0.60 0.28 Leu 22 . A B . . . . −1.60 0.80 . . . −0.60 0.28 Pro 23 . AB . . . . −1.64 0.87 . * . −0.60 0.28 Val 24 . . B . . . . −1.32 1.01. * . −0.40 0.25 Pro 25 . . B . . . . −1.08 0.33 . . . −0.10 0.60 Ala 26. . B B . . . −1.12 0.07 . . . −0.30 0.38 Val 27 . . B B . . . −0.900.29 * * . −0.30 0.38 Arg 28 . . B B . . . −0.69 0.14 * * . −0.30 0.25Gly 29 . . B B . . . −0.14 −0.29 * * . 0.30 0.43 Val 30 . . B B . . .−0.14 −0.30 * * . 0.30 0.83 Ala 31 . . B B . . . 0.13 −0.51 * * . 0.600.66 Glu 32 . . B . . . . 0.74 −0.03 * * F 0.65 0.96 Thr 33 . . B . . T. 0.42 0.30 * * F 0.40 2.02 Pro 34 . . . . T T . 0.48 0.09 * * F 0.803.10 Thr 35 . . . . T T . 1.44 0.50 * . F 0.50 1.88 Tyr 36 . . . . . T C2.03 0.50 * . . 0.15 2.55 Pro 37 . . . . T . . 1.44 0.01 * . . 0.45 2.76Trp 38 . A . . . . C 1.76 0.09 * . . 0.05 1.93 Arg 39 . A B . . . . 1.66−0.40 * . F 0.60 2.13 Asp 40 . A . . . . C 1.62 −0.67 * . F 1.10 1.99Ala 41 . A . . . . C 1.87 −0.67 * * F 1.10 1.87 Glu 42 . A . . . . C2.19 −1.59 * * F 1.10 1.66 Thr 43 . A . . T . . 1.67 −1.59 * . F 1.301.94 Gly 44 . A . . T . . 0.70 −0.90 * . F 1.30 1.59 Glu 45 . A . . T .. 0.03 −0.76 * * F 1.15 0.68 Arg 46 . A . . T . . 0.03 −0.19 * . F 0.850.25 Leu 47 . A B . . . . 0.03 −0.17 * . . 0.30 0.26 Val 48 . A B . . .. −0.32 −0.20 . . . 0.30 0.26 Cys 49 . A B . . . . −0.19 0.37 . . .−0.30 0.07 Ala 50 . A B . . . . −0.40 0.80 . . . −0.60 0.13 Gln 51 . A B. . . . −0.86 0.54 . . . −0.60 0.28 Cys 52 . A B . . . . −0.36 0.33 . .. −0.30 0.51 Pro 53 . . . . . T C −0.20 0.24 . . F 0.45 0.73 Pro 54 . .. . T T . −0.39 0.53 . . F 0.35 0.36 Gly 55 . . . . T T . 0.20 0.77 * .F 0.35 0.50 Thr 56 . . B . . T . 0.31 0.60 . . F −0.05 0.56 Phe 57 . . BB . . . 0.77 0.17 * . F −0.15 0.71 Val 58 . . B B . . . 0.31 0.17 * . .0.19 1.12 Gln 59 . . B B . . . 0.63 0.31 * . F 0.53 0.41 Arg 60 . . B .. T . 1.09 −0.17 * . F 1.87 0.94 Pro 61 . . B . . T . 1.40 −0.96 * . F2.66 2.47 Cys 62 . . . . T T . 1.80 −1.60 * . F 3.40 2.38 Arg 63 . . . .T T . 2.44 −1.61 * . F 3.06 1.63 Arg 64 . . . . T . . 2.13 −1.19 * . F2.77 1.63 Asp 65 . . . . T . . 1.71 −1.13 * . F 2.68 4.39 Ser 66 . . . .T T . 1.26 −1.21 . . F 2.79 3.24 Pro 67 . . . . T T . 1.58 −0.64 . . F2.55 0.89 Thr 68 . . . . T T . 1.26 −0.21 . . F 2.50 0.52 Thr 69 . . . .T T . 0.48 0.21 . . F 1.65 0.61 Cys 70 . . . . T . . 0.27 0.40 . . F1.14 0.21 Gly 71 . . . . T T . 0.36 0.40 . * F 1.33 0.22 Pro 72 . . . .T T . 0.68 0.34 . * F 1.62 0.24 Cys 73 . . . . . T C 0.96 −0.14 * . F2.01 0.88 Pro 74 . . . . . T C 1.02 −0.21 * . F 2.40 1.21 Pro 75 . . . .T T . 1.38 0.11 * * F 1.76 1.23 Arg 76 . . . . T T . 1.72 0.17 * * F1.52 3.30 His 77 . . B . . T . 1.23 0.00 * * F 0.88 3.70 Tyr 78 . . B .. T . 1.61 0.36 * * . 0.49 2.07 Thr 79 . . B . . . . 1.82 0.84 * * .−0.25 1.11 Gln 80 . . B . . . . 1.79 1.24 * * . −0.25 1.31 Phe 81 . . .. T . . 0.87 1.50 * * . 0.15 1.31 Trp 82 . . . . T . . 0.90 1.43 * . .0.00 0.75 Asn 83 . A . . T . . 1.26 0.94 * . . −0.20 0.75 Tyr 84 . A . .T . . 0.90 0.54 * * . −0.05 1.70 Leu 85 . A . . T . . 1.01 0.33 * * .0.38 0.87 Glu 86 . A . . T . . 1.47 −0.59 * * . 1.71 1.05 Arg 87 . A . .T . . 1.09 −0.23 . * . 1.69 1.05 Cys 88 . . . . T T . 1.09 −0.41 . * .2.22 0.69 Arg 89 . . . . T T . 0.48 −0.70 . * . 2.80 0.64 Tyr 90 . . . .T T . 0.48 −0.06 . * . 2.22 0.24 Cys 91 . . . . T T . −0.19 0.63 . * .1.04 0.37 Asn 92 . . B B . . . −0.64 0.63 . * . −0.04 0.10 Val 93 . . BB . . . 0.02 1.06 . * . −0.02 0.06 Leu 94 . . B B . . . 0.02 0.30 . . .0.30 0.21 Cys 95 . . B . . T . 0.27 −0.27 . . . 1.60 0.25 Gly 96 . . . .. T C 0.93 −0.67 . . F 2.55 0.59 Glu 97 . . . . . T C 0.93 −1.31 . . F3.00 1.24 Arg 98 A . . . . T . 1.20 −2.00 . * F 2.50 4.00 Glu 99 A A . .. . . 2.12 −2.07 . * F 1.80 4.08 Glu 100 A A . . . . . 2.20 −2.50 . * F1.50 4.61 Glu 101 A A . . . . . 1.88 −2.00 . * F 1.20 2.38 Ala 102 A A .. . . . 1.84 −1.43 . . F 0.75 0.74 Arg 103 A A . . . . . 1.14 −0.93 . .. 0.60 0.58 Ala 104 A A . . . . . 0.83 −0.43 . * . 0.30 0.34 Cys 105 A A. . . . . 0.80 0.06 . * . −0.30 0.48 His 106 A A . . . . . 0.80 0.06 * *. −0.30 0.34 Ala 107 . A . . T . . 1.50 0.46 * * . −0.20 0.53 Thr 108 .A . . T . . 0.80 −0.04 * * . 0.85 1.95 His 109 . A . . T . . 0.72−0.11 * . . 1.13 1.45 Asn 110 . A . . T . . 1.50 −0.04 * . . 1.26 0.77Arg 111 . A . . T . . 0.87 −0.54 . * . 1.99 1.04 Ala 112 . A . . T . .1.57 −0.46 . * . 1.82 0.41 Cys 113 . . . . T T . 1.57 −0.96 . * . 2.800.50 Arg 114 . . B . . T . 1.26 −0.87 . * . 2.12 0.37 Cys 115 . . . . TT . 0.56 −0.44 * * . 1.94 0.36 Arg 116 . . . . T T . −0.26 −0.16 . * .1.66 0.58 Thr 117 . A . B T . . −0.26 0.06 . * F 0.53 0.26 Gly 118 . A .B T . . 0.38 0.56 . * . −0.20 0.49 Phe 119 . A B B . . . −0.32 0.49 . *. −0.60 0.34 Phe 120 . A B B . . . −0.00 0.99 . * . −0.60 0.24 Ala 121 .A B B . . . −0.81 0.93 . * . −0.60 0.24 His 122 . A . . . . C −1.17 1.29. * . −0.40 0.24 Ala 123 . A . . . . C −1.63 1.07 . * . −0.40 0.15 Gly124 . A . . T . . −0.93 0.97 . * . −0.20 0.12 Phe 125 . A . . T . .−0.27 0.47 . . . −0.20 0.15 Cys 126 . A . . T . . −0.27 0.47 . * . −0.200.20 Leu 127 . A B . . . . −0.53 0.47 . . . −0.60 0.21 Glu 128 . A B . T. . −0.61 0.43 . . . −0.20 0.32 His 129 . . . . T T . −0.48 0.21 . . .0.50 0.32 Ala 130 . . . . T T . 0.01 0.07 . . . 0.63 0.61 Ser 131 . . .. T T . 0.33 −0.19 . . . 1.36 0.54 Cys 132 . . . . . T C 0.56 0.24 . . .0.69 0.39 Pro 133 . . . . . T C 0.21 0.24 . . F 0.97 0.39 Pro 134 . . .. T T . −0.61 0.17 . . F 1.30 0.29 Gly 135 . . . . T T . −0.91 0.43 . .F 0.87 0.40 Ala 136 . . B . . T . −1.20 0.54 . . . 0.19 0.18 Gly 137 . .B B . . . −0.74 0.61 . . . −0.34 0.12 Val 138 . . B B . . . −0.88 0.61 .. . −0.47 0.19 Ile 139 . . B B . . . −0.67 0.61 . . . −0.60 0.18 Ala 140. . B . . T . −0.62 0.11 . . . 0.10 0.32 Pro 141 . . B . . T . −0.320.07 . . F 0.25 0.58 Gly 142 . . . . . T C −0.57 0.34 * * F 0.45 0.86Glu 143 . . . . . T C 0.40 0.16 * * F 0.45 0.86 Ser 144 . . B . . . .0.94 −0.34 * * F 0.80 1.10 Trp 145 . . . . T . . 1.19 −0.34 * * F 1.201.10 Ala 146 . . B . . T . 0.81 −0.34 * * F 0.85 0.63 Arg 147 . . . . TT . 0.94 0.16 * * F 0.65 0.47 Gly 148 . . . . T T . 1.06 0.20 . * F 0.650.69 Gly 149 . . . . . T C 1.06 −0.71 . . F 1.84 1.35 Ala 150 . . . . .. C 1.00 −0.83 . . F 1.83 0.92 Pro 151 . . . . . . C 1.24 −0.40 . * F1.87 0.92 Arg 152 . . . . T T . 1.24 −0.40 . . F 2.61 0.92 Ser 153 . . .. T T . 1.70 −0.83 * . F 3.40 1.78 Gly 154 . . . . T T . 1.38 −1.33 * *F 3.06 2.26 Gly 155 . . . . T T . 1.62 −1.19 * * F 2.57 0.62 Arg 156 . .. . T . . 1.94 −0.76 * * F 2.26 0.46 Arg 157 . . . . T . . 1.49−1.14 * * F 2.15 0.90 Cys 158 . . B . . . . 1.79 −1.14 * * F 1.64 0.90Gly 159 . . . . T T . 1.28 −1.17 * * F 2.47 0.80 Arg 160 . . B . . T .1.03 −0.53 * * F 2.30 0.30 Gly 161 . . B . . T . 0.58 −0.03 * * F 1.770.57 Gln 162 . . B . . T . 0.26 −0.17 * * F 1.54 0.57 Val 163 . . B . .. . 0.62 −0.17 . * F 1.11 0.45 Ala 164 . . B . . . . 0.16 0.21 . * F0.28 0.61 Gly 165 . . B . . T . −0.54 0.47 . * F −0.05 0.29 Pro 166 . .B . . T . −0.41 0.57 . . F −0.05 0.40 Ser 167 . . . . . T C −0.80 0.36 .. F 0.45 0.61 Leu 168 . . B . . T . −0.33 0.29 . . . 0.10 0.78 Ala 169 .. B . . . . −0.13 0.29 . . . −0.10 0.65 Pro 170 . . B . . . . −0.18 0.29. . . −0.10 0.62

Additional preferred nucleic acid fragments of the present inventioncomprise, or alternatively consist of, nucleic acid molecules encodingone or more epitope-bearing portions of TNFR-6α and/or TNFR-6β. Inparticular, such nucleic acid fragments of the present invention includenucleic acid molecules encoding: a polypeptide comprising, oralternatively consisting of, amino acid residues from about Phe-57 toabout Thr-117, from about Cys-132 to about Thr-175, from about Gly-185to about Thr-194, from about Val-205 to about Asp-217, from aboutPro-239 to about Leu-264, and/or from about Ala-283 to about Pro-298 inSEQ ID NO:2. In additional embodiments, nucleic acid fragments of thepresent invention comprise, or alternatively consist of nucleic acidmolecules encoding one or more epitpope bearing portions of TNFR-6β fromabout Ala-31 to about Thr-46, from about Phe-57 to about Gln-80, fromabout Glu-86 to about His-106, from about Thr-108 to about Phe-119, fromabout His-129 to about Val-138, and/or from about Gly-142 to aboutPro-166 in SEQ ID NO:4. In this context “about” includes theparticularly recited ranges and rangers larger or smaller by several (5,4, 3, 2, or 1) amino acids at either terminus or at both termini. Thesepolypeptide fragments have been determined to bear antigenic epitopes ofthe TNFR-6α and TNFR-6β polypeptides respectively, by the analysis ofthe Jameson-Wolf antigenic index, as shown in FIGS. 4 and 5, above.Further, polypeptide fragments which bear antigenic epitopes of TNFR-6αand/or TNFR-6β may be easily determined by one of skill in the art usingthe above-described analysis of the Jameson-Wolf antigenic index, asshown in FIGS. 4 and 5. Methods for determining other suchepitope-bearing portions of TNFR-6α and/or TNFR-6β are described indetail below.

In specific embodiments, the nucleic acids of the invention are lessthan 100000 kb, 50000 kb, 10000 kb, 1000 kb, 500 kb, 400 kb, 350 kb, 300kb, 250 kb, 200 kb, 175 kb, 150 kb, 125 kb, 100 kb, 75 kb, 50 kb, 40 kb,30 kb, 25 kb, 20 kb, 15 kb, 10 kb, 7.5 kb, or 5 kb in length.

In further embodiments, nucleic acids of the invention comprise at least15, at least 30, at least 50, at least 100, or at least 250, at least500, or at least 1000 contiguous nucleotides of TNFR coding sequence,but consist of less than or equal to 1000 kb, 500 kb, 250 kb, 200 kb,150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kbof genomic DNA that flanks the 5′ or 3′ coding nucleotide sequence setforth in FIG. 1 (SEQ ID NO:1) or FIGS. 2A-B (SEQ ID NO:3). In furtherembodiments, nucleic acids of the invention comprise at least 15, atleast 30, at least 50, at least 100, or at least 250, at least 500, orat least 1000 contiguous nucleotides of TNFR coding sequence, but do notcomprise all or a portion of any TNFR intron. In another embodiment, thenucleic acid comprising TNFR coding sequence does not contain codingsequences of a genomic flanking gene (i.e., 5′ or 3′ to the TNFR gene inthe genome). In other embodiments, the nucleic acids of the invention donot contain the coding sequence of more than 1000, 500, 250, 100, 50,25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising, or alternatively consisting of, a polynucleotidewhich hybridizes under stringent hybridization conditions to a portionof the polynucleotide in a nucleic acid molecule of the inventiondescribed above, for instance, the cDNA contained in the plasmiddeposited as ATCC Deposit No. 97810 or 97809, or a fragment of thepolynucleotide sequence disclosed in FIG. 1 and/or FIGS. 2A-B. By“stringent hybridization conditions” is intended overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 (e.g., 50) nt of the reference polynucleotide. These haveuses that include, but are not limited to, as diagnostic probes andprimers as discussed above and in more detail below.

By a portion of a polynucleotide of “at least about 20 nt in length,”for example, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., a depositedcDNA or a nucleotide sequence as shown in FIG. 1 or 2 (SEQ ID NO:1 or3)). In this context “about” includes the particularly recited size, andthose sizes that are larger or smaller by several (5, 4, 3, 2, or 1)nucleotides, at either terminus or at both termini. Of course, apolynucleotide which hybridizes only to a poly A sequence (such as the3′ terminal poly(A) tract of a TNFR cDNA, or to a complementary stretchof T (or U) residues, would not be included in a polynucleotide of theinvention used to hybridize to a portion of a nucleic acid of theinvention, since such a polynucleotide would hybridize to any nucleicacid molecule containing a poly (A) stretch or the complement thereof(e.g., practically any double-stranded cDNA clone that has beengenerated using oligo dT as a primer).

As indicated, nucleic acid molecules of the present invention whichencode a TNFR polypeptide may include, but are not limited to, thoseencoding the amino acid sequence of the mature polypeptide, by itself;and the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about 26-35 amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro-protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences.

Also encoded by nucleic acids of the invention are the above proteinsequences together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; anadditional coding sequence which codes for additional amino acids, suchas those which provide additional functionalities.

Thus, the sequence encoding the polypeptide may be fused to a markersequence, such as a sequence encoding a peptide which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37: 767 (1984).As discussed below, other such fusion proteins include a TNFR-6α orTNFR-6β fused to Fc at the N— or C-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of a TNFR polypeptide. Variants may occur naturally, such asa natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques which include, but are not limited tooligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res.13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),cassette mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)),restriction selection mutagenesis (see e.g., Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)).

Thus, the invention also encompasses TNFR variants (e.g., derivativesand analogs) that have one or more amino acid residues deleted, added,or substituted to generate TNFR polypeptides that are better suited forexpression, scale up, etc., in the host cells chosen. For example,cysteine residues can be deleted or substituted with another amino acidresidue in order to eliminate disulfide bridges; N-linked glycosylationsites can be altered or eliminated to achieve, for example, expressionof a homogeneous product that is more easily recovered and purified fromyeast hosts which are known to hyperglycosylate N-linked sites. To thisend, a variety of amino acid substitutions at one or both of the firstor third amino acid positions on any one or more of the glycosylationrecognition sequences in the TNFR polypeptides of the invention, and/oran amino acid deletion at the second position of any one or more suchrecognition sequences will prevent glycosylation of the TNFR at themodified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J5(6):1193-97). Additionally, one or more of the amino acid residues ofthe polypeptides of the invention (e.g., arginine and lysine residues)may be deleted or substituted with another residue to eliminateundesired processing by proteases such as, for example, furins orkexins. For example, polypeptides of the invention containing carboxyterminal TNFR polypeptide sequences may have the amino acid residuecorresponding to the arginine residue at position 290 and/or 295 of SEQID NO:2 deleted or substituted with another residue.

Variants of the invention include those produced by nucleotidesubstitutions, deletions or additions. The substitutions, deletions oradditions may involve one or more nucleotides. The variants may bealtered in coding regions, non-coding regions, or both. Alterations inthe coding regions may produce conservative or non-conservative aminoacid substitutions, deletions or additions. Especially preferred amongthese are silent substitutions, additions and deletions, which do notalter the properties and activities of the TNFR polypeptide or portionsthereof. Also especially preferred in this regard are conservativesubstitutions.

Highly preferred are nucleic acid molecules encoding a mature proteinhaving an amino acid sequence shown in SEQ ID NOS:2 and 4 or the matureTNFR polypeptide sequences encoded by the cDNA clone contained in theplasmid deposited as ATCC Deposit No. 97810 or ATCC Deposit No. 97809.

Further embodiments include an isolated nucleic acid moleculecomprising, or alternatively consisting of, a polynucleotide having anucleotide sequence at least 90% identical, and more preferably at least80%, 85%, 90%, 92%, or 95%, 96%, 97%, 98% or 99% identical to apolynucleotide selected from the group consisting of: (a) a nucleotidesequence encoding a TNFR polypeptide having the complete amino acidsequence in SEQ ID NO:2 or 4, or as encoded by the cDNA clone containedin the plasmid deposited as ATCC Deposit No. 97810 or 97809; (b) anucleotide sequence encoding a mature TNFR polypeptide having an aminoacid sequence at positions 31-300 or 31-170 in SEQ ID NO:2 or 4,respectively, or as encoded by the cDNA clone contained in the plasmiddeposited as ATCC Deposit No. 97810 or 97809; (c) a nucleotide sequenceencoding a soluble extracellular domain of a TNFR polypeptide having theamino acid sequence at positions 31-283 and 31-166 of SEQ ID NOS:2 and4, respectively; (d) a nucleotide sequence encoding a fragment of theTNFR polypeptide having the complete amino acid sequence in SEQ ID NO:2or 4, or as encoded by the cDNA clone contained in the plasmid depositedas ATCC Deposit No. 97810 or 97809, wherein the fragment has TNFR-6αand/or TNFR-6β functional activity; and (e) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c) or (d)above. Polypeptides encoded by the polynucleotides are also encompassedby the invention.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 80%, 85%, 90%, 92%, or95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequencesin (a), (b), (c), (d), or (e), above, or a polynucleotide whichhybridizes under stringent hybridization conditions to a polynucleotidein (a), (b), (c), (d), or (e), above. This polynucleotide whichhybridizes does not hybridize under stringent hybridization conditionsto a polynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues. An additional nucleic acid embodiment ofthe invention relates to an isolated nucleic acid molecule comprising,or alternatively consisting of, a polynucleotide which encodes the aminoacid sequence of an epitope-bearing portion of a TNFR polypeptide havingan amino acid sequence in (a), (b), (c), (d), or (e), above.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a TNFRpolypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the TNFRpolypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 80%, 85%, 90%, 92%, or 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. The reference sequence may be theentire TNFR-6α and/or TNFR -6β encoding sequence shown in FIG. 1 (SEQ IDNO:1 and 2) and FIGS. 2A-B (SEQ ID NO:3 and 4) or any fragment, variant,derivative or analog thereof, as described herein.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, anucleotide sequence shown in FIG. 1 or 2, or to the nucleotides sequencecontained in one or both of the deposited cDNA clones can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2:482-489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed. The reference (query) sequence may be the entire TNFR encodingnucleotide sequence shown in FIG. 1 (SEQ ID NO:1), FIGS. 2A-B (SEQ IDNO:3) or any TNFR-6α and/or TNFR-6β polynucleotide fragment (e.g,. apolynucleotide encoding the amino acid sequence of any of the N or Cterminal deletions described herein), variant, derivative or analog, asdescribed herein.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions, a manual correction ismade to the results to take into consideration the fact that the FASTDBprogram does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.A determination of whether a nucleotide is matched/aligned is determinedby results of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This corrected score is what is used for the purposes ofthis embodiment. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score. For example, a 90 basesubject sequence is aligned to a 100 base query sequence to determinepercent identity. The deletions occur at the 5′ end of the subjectsequence and therefore, the FASTDB alignment does not show amatched/alignment of the first 10 bases at 5′ end. The 10 unpaired basesrepresent 10% of the sequence (number of bases at the 5′ and 3′ ends notmatched/total number of bases in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 bases were perfectly matched the finalpercent identity would be 90%. In another example, a 90 base subjectsequence is compared with a 100 base query sequence. This time thedeletions are internal deletions so that there are no bases on the 5′ or3′ of the subject sequence which are not matched/aligned with the query.In this case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only bases 5′ and 3′ of the subject sequencewhich are not matched/aligned with the query sequence are manuallycorrected for. No other manual corrections are made for the purposes ofthis embodiment.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequenceshown in FIG. 1 or 2 (SEQ ID NO:1 or 3), to the nucleic acid sequence ofa deposited cDNA and/or to a nucleic acid sequence otherwise disclosedherein (e.g., encoding polypeptide having the amino acid sequence of a Nand/or C terminal deletion disclosed herein, such as, for example, anucleic acid molecule encoding amino acids Val-30 to His-300 of SEQ IDNO:2), irrespective of whether they encode a polypeptide having TNFRfunctional activity. This is because even where a particular nucleicacid molecule does not encode a polypeptide having TNFR functionalactivity, one of skill in the art would still know how to use thenucleic acid molecule, for instance, as a hybridization probe or apolymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving TNFR functional activity include, inter alia, (1) isolating aTNFR gene or allelic variants thereof in a cDNA library; (2) in situhybridization (e.g., “FISH”) to metaphase chromosomal spreads to provideprecise chromosomal location of the TNFR gene, as described in Verma etal., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York (1988); and Northern Blot analysis for detecting TNFR mRNAexpression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequenceshown in FIG. 1 or 2 (SEQ ID NOS:1 or 3) or to the nucleic acid sequenceof the cDNA clone contained in the plasmid deposited as ATCC Deposit No.97810 or ATCC Deposit No. 97809, and/or to a nucleic acid sequenceotherwise disclosed herein (e.g., encoding polypeptide having the aminoacid sequence of a N and/or C terminal deletion disclosed herein), whichdo, in fact, encode polypeptides having TNFR (i.e., TNFR-6α and/orTNFR-6β) protein functional activity. By “a polypeptide having TNFRfunctional activity” is intended polypeptides exhibiting activitysimilar, but not necessarily identical, to an activity of a TNFR-6αand/or TNFR-6β protein of the invention (e.g., complete (full-length),mature, and extracellular domain as measured, for example, in aparticular immunoassay or biological assay. For example, TNFR-6α and/orTNFR-6β activity can be measured by determining the ability of a TNFR-6αand/or TNFR-6β polypeptide to bind a TNFR-6α and/or -6β ligand (e.g.,Fas Ligand and/or AIM-II (International application publication numberWO 97/34911, published Sep. 25, 1997). In another example, TNFR-6αand/or TNFR-6β functional activity is measured by determining theability of a polypeptide, such as cognate ligand which is free orexpressed on a cell surface, to induce apoptosis.

The TNF family ligands induce various cellular responses by binding toTNF-family receptors, including the TNFR-6α and TNFR-6β of the presentinvention. Cells which express the TNFR proteins are believed to have apotent cellular response to TNFR-I receptor ligands including Blymphocytes (CD19+), both CD4 and CD8+ T lymphocytes, monocytes andendothelial cells. By a “cellular response to a TNF-family ligand” isintended any genotypic, phenotypic, and/or morphological change to acell, cell line, tissue, tissue culture or patient that is induced by aTNF-family ligand. As indicated, such cellular responses include notonly normal physiological responses to TNF-family ligands, but alsodiseases associated with increased cell proliferation or the inhibitionof increased cell proliferation, such as by the inhibition of apoptosis.

Screening assays for the forgoing are known in the art. One suchscreening assay involves the use of cells which express the receptor(for example, transfected CHO cells) in a system which measuresextracellular pH changes caused by receptor activation, for example, asdescribed in Science 246:181-296 (October 1989). For example, aTNF-family ligand may be contacted with a cell which expresses themature form of the receptor polypeptide of the present invention and asecond messenger response, e.g., signal transduction or pH changes, maybe measured to determine whether the TNFR polypeptide is active.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the cDNA clonedeposited as ATCC Deposit No. 97810 or 97809, the nucleic acid sequenceshown in FIG. 1 or 2 (SEQ ID NO:1 and 3), or fragments thereof, willencode a polypeptide “having TNFR protein functional activity.” In fact,since degenerate variants of these nucleotide sequences all encode thesame polypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving TNFR protein functional activity. This is because the skilledartisan is fully aware of amino acid substitutions that are either lesslikely or not likely to significantly effect protein function (e.g.,replacing one aliphatic amino acid with a second aliphatic amino acid),as further described below.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatednucleic acid molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, or which areotherwise engineered to produce the polypeptides of the invention, andthe production of TNFR polypeptides, or fragments thereof, byrecombinant techniques.

In one embodiment, the polynucleotides of the invention are joined to avector (e.g., a cloning or expression vector). The vector may be, forexample, a phage, plasmid, viral or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells. Generally, a plasmid vector is introduced in aprecipitate, such as a calcium phosphate precipitate, or in a complexwith a charged lipid. If the vector is a virus, it may be packaged invitro using an appropriate packaging cell line and then transduced intohost cells.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The expression constructs will furthercontain sites for transcription initiation, termination and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the transcripts expressed by the constructs will preferablyinclude a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated. The heterologous structuralsequence is assembled in appropriate phase with translation initiationand termination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, for example, stabilization or simplifiedpurification of expressed recombinant product.

In one embodiment, the DNA of the invention is operatively associatedwith an appropriate heterologous regulatory element (e.g., promoter orenhancer), such as, the phage lambda PL promoter, the E. coli lac, trp,phoA, and tac promoters, the SV40 early and late promoters and promotersof retroviral LTRS, to name a few. Other suitable promoters will beknown to the skilled artisan.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,glutamine synthase, G418 or neomycin resistance for eukaryotic cellculture and tetracycline, kanamycin or ampicillin resistance genes forculturing in E. coli and other bacteria.

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. The availability of drugswhich inhibit the function of the enzymes encoded by these selectablemarkers allows for selection of cell lines in which the vector sequenceshave been amplified after integration into the host cell's DNA. Anadvantage of glutamine synthase based vectors are the availabilty ofcell lines (e.g., the murine myeloma cell line, NS0) which are glutaminesynthase negative. Vectors containing glutamine synthase can also beamplified in glutamine synthase expressing cells (e.g. Chinese HamsterOvary (CHO) cells) by providing additional inhibitor to prevent thefunctioning of the endogenous gene. A glutamine synthase expressionsystem and components thereof are detailed in PCT publications:WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657 which arehereby incorporated in their entireties by reference herein.Additionally, glutamine synthase expression vectors that may be usedaccording to the present invention are commercially available fromsuppliers including, for example, Lonza Biologics, Inc. (Portsmouth,N.H.). Expression and production of monoclonal antibodies using a GSexpression system in murine myeloma cells is described in Bebbington etal., Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol.Prog. 11:1 (1995) which are herein incorporated by reference.

Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS, 293 and Bowes melanoma cells; and plant cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

The host cell can be a higher eukaryotic cell, such as a mammalian cell(e.g., a human derived cell), or a lower eukaryotic cell, such as ayeast cell, or the host cell can be a prokaryotic cell, such as abacterial cell. The host strain may be chosen which modulates theexpression of the inserted gene sequences, or modifies and processes thegene product in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thusexpression of the genetically engineered polypeptide may be controlled.Furthermore, different host cells have characteristics and specificmechanisms for the translational and post-translational processing andmodification (e.g., phosphorylation, cleavage) of proteins. Appropriatecell lines can be chosen to ensure the desired modifications andprocessing of the foreign protein expressed. Selection of appropriatevectors and promoters for expression in a host cell is a well knownprocedure and the requisite techniques for expression vectorconstruction, introduction of the vector into the host and expression inthe host are routine skills in the art.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium, and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice. As a representative, but nonlimiting example,useful expression vectors for bacterial use can comprise a selectablemarker and bacterial origin of replication derived from commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017). Such commercial vectors include, forexample, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1(Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sectionsare combined with an appropriate promoter and the structural sequence tobe expressed. Among vectors preferred for use in bacteria include pHE4-5(ATCC Accession No. 209311; and variations thereof), pQE70, pQE60 andpQE-9, available from QIAGEN, Inc., supra; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,pMSG and pSVL available from Pharmacia. Other suitable vectors will bereadily apparent to the skilled artisan.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman (Cell23:175 (1981)), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, NSO HeLa and BHK cell lines.NSO cell lines are particularly suitable host cells for transformationwith polynucleotides and expression vectors of the invention. Mammalianexpression vectors will comprise an origin of replication, a suitablepromoter and enhancer, and also any necessary ribosome binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking nontranscribed sequences. DNAsequences derived from the SV40 splice, and polyadenylation sites may beused to provide the required nontranscribed genetic elements.

Introduction of the vector construct into the host cell can be effectedby techniques known in the art which include, but are not limited to,calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., TNFR coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with TNFR polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous TNFR polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous TNFR polynucleotide sequences via homologous recombination(see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; Internationalapplication publication number WO 96/29411, published Sep. 26, 1996;International application publication number WO 94/12650, published Aug.4, 1994; Koller et al., Proc. Natl. Acad Sci. USA 86:8932-8935 (1989);and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of eachof which are incorporated by reference in their entireties).

The host cells described infra can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, cell-free translation systems can also be employed toproduce the polypeptides of the invention using RNAs derived from theDNA constructs of the present invention.

The polypeptide of the invention may be expressed or synthesized in amodified form, such as a fusion protein (comprising the polypeptidejoined via a peptide bond to a heterologous protein sequence (of adifferent protein), e.g., the signal peptide of CK-beta8 (amino acids−21 to −1 of the CK-beta8 sequence disclosed in published PCTapplication PCT/US95/09058; filed Jun. 23, 1995) or the signal peptideof stanniocalcin (See ATCC Accession No. 75652, deposited Jan. 25,1994)), and may include not only secretion signals, but also additionalheterologous functional regions. Such a fusion protein can be made byligating polynucleotides of the invention and the desired nucleic acidsequence encoding the desired amino acid sequence to each other, bymethods known in the art, in the proper reading frame, and expressingthe fusion protein product by methods known in the art. Alternatively,such a fusion protein can be made by protein synthetic techniques, e.g.,by use of a peptide synthesizer. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art.

In one embodiment, polynucleotides encoding TNFR-6 alpha and/or TNFR-6beta polypeptides of the invention may be fused to signal sequenceswhich will direct the localization of a protein of the invention toparticular compartments of a prokaryotic or eukaryotic cell and/ordirect the secretion of a protein of the invention from a prokaryotic oreukaryotic cell. For example, in E. coli, one may wish to direct theexpression of the protein to the periplasmic space. Examples of signalsequences or proteins (or fragments thereof) to which the polypeptidesof the invention may be fused in order to direct the expression of thepolypeptide to the periplasmic space of bacteria include, but are notlimited to, the pelB signal sequence, the maltose binding protein (MBP)signal sequence, MBP, the ompA signal sequence, the signal sequence ofthe periplasmic E. coli heat-labile enterotoxin B-subunit, the signalsequence of chemokine-beta-8, and the signal sequence of alkalinephosphatase. Several vectors are commercially available for theconstruction of fusion proteins which will direct the localization of aprotein, such as the pMAL series of vectors (particularly the pMAL-pseries) available from New England Biolabs. In a specific embodiment,polynucleotides encoding TNFR-6 alpha and/or TNFR-6 beta polypeptides ofthe invention may be fused to the pelB pectate lyase signal sequence toincrease the efficiency of expression and purification of suchpolypeptides in Gram-negative bacteria. See, U.S. Pat. Nos. 5,576,195and 5,846,818, the contents of which are herein incorporated byreference in their entireties.

Examples of signal peptides that may be fused to a polypeptide of theinvention in order to direct its secretion in mammalian cells include,but are not limited to, the MPIF-1 signal sequence (amino acids 1-21 ofGenBank Accession number AAB51134), the stanniocalcin signal sequence(MLQNSAVLLLLVISASA, SEQ ID NO:29), and a consensus signal sequence(MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:30). A suitable signal sequence thatmay be used in conjunction with baculoviral expression systems is thegp67 signal sequence, (amino acids 1-19 of GenBank Accession NumberAAA72759).

A preferred fusion protein comprises a heterologous region fromimmunoglobulin that is useful to stabilize and purify proteins. Forexample, EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusionproteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). Inpreferred embodiments, the IgG1 m(f) allele is used to generate Fcfusion proteins of the invention. In other embodiments, the IgG1 m(f)allele in which the cysteine residue at position 220 in the hinge regionof the constant region is substituted with a serine amino acid residueis used. Alternatively, for some uses it would be desirable to be ableto delete the Fc part after the fusion protein has been expressed,detected and purified in the advantageous manner described. This is thecase when Fc portion proves to be a hindrance to use in therapy anddiagnosis, for example when the fusion protein is to be used as antigenfor immunizations. As an example, an enterokinase cleavage site betweenthe protein of the invention and the fusion moiety (e.g. Fc constantregion) may be engineered. Subsequently, the protein of the inventionmay be separated from the fusion moiety by digestion with enterokinase.In drug discovery, for example, human proteins, such as hIL-5 has beenfused with Fc portions for the purpose of high-throughput screeningassays to identify antagonists of hIL-5. See, D. Bennett et al., J.Molecular Recognition 8:52-58 (1995) and K. Johanson et al., J. Biol.Chem. 270:9459-9471 (1995). In another example, preferred fusionproteins of the invention comprise a portion of an immunoglobulin lightchain (i.e., a portion of a kappa or lambda light chain). In specificembodiments the fusion proteins of the invention comprise a portion ofthe constant region of a kappa or lambda light chain.

Polypeptides of the invention (including antibodies of the invention,see below) may also be fused to albumin (including but not limited torecombinant human serum albumin (HSA) (see, e.g., U.S. Pat. No.5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No.5,766,883, issued Jun. 16, 1998, herein incorporated by reference intheir entirety)), resulting in chimeric polypeptides. In a preferredembodiment, polypeptides (including antibodies) of the present invention(including fragments or variants thereof) are fused with the mature formof human serum albumin (i.e., amino acids 1-585 of human serum albuminas shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is hereinincorporated by reference in its entirety. In another preferredembodiment, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) are fused with polypeptidefragments comprising, or alternatively consisting of, amino acidresidues 1-z of human serum albumin, where z is an integer from 369 to419, as described in U.S. Pat. No. 5,766,883 herein incorporated byreference in its entirety. Polypeptides and/or antibodies of the presentinvention (including fragments or variants thereof) may be fused toeither the N— or C-terminal end of the heterologous protein (e.g.,immunoglobulin Fc polypeptide or human serum albumin polypeptide). Suchhuman serum albumin TNFR-6 alpha and TNFR-6 beta fusion proteins may beused therapeutically in accordance with the invention, in the samemanner as, for example, the TNFR-6α- and TNFR-6β-Fc fusion proteinsdescribed herein, below (see, e.g., Examples 22 and 23).

Other fusion proteins of the invention include fusion of TNFR-6 alpha orTNFR-6 beta, or even TNFR-6 alpha-Fc fusion protein, TNFR-6 beta-Fcfusion protein, TNFR-6 alpha-HSA fusion protein, or TNFR-6 beta-HSAfusion protein, fused to glucoamylase (e.g., GenBank Accession NumberP23176 or P04064). Nucleic acids encoding such fusion proteins operablyassociated with appropriate regulatory sequences and/or selectablemarkers may be expressed in fungal cells including, for example,Saccharomyces species such as S. cerevisiae, Aspergillus species such asA. niger and Chrysosporium species such as C. lucknowense.

Exemplary fragments of TNFR-6 alpha, that may be fused to a heterologouspolypeptide, for example, immunoglobulin Fc domain or human serumalbumin include, but are not limited to, amino acid residues 1-299,1-300, 23-300, 34-300, 30-300, 1-195, 1-221, 1-254, 1-271, 35-300,42-300, 47-300, and 48-300 SEQ ID NO:2. In preferred embodiments, aminoacid residues 30-300 of SEQ ID NO:2 are fused to an immunoglobulin Fcdomain or human serum albumin.

In specific embodiments, the present invention provides TNFR-6 alphaexpression constructs for expressing TNFR-6 alpha or fragments, variantsor fusion proteins thereof, in which the polynucleotide encoding theTNFR-6 alpha polypeptide comprises exons 1, 2, and 3 of TNFR-6 alpha aswell as the intervening introns, see for example SEQ ID NO:27, whereinexon I consists of nucleotides 425-560 of SEQ ID NO:27 and exon 2consists of nucleotides 756-1512 of SEQ ID NO:27. In particularembodiments, the above expression constructs comprising TNFR-6 alphaexons and introns may also be designed so that the TNFR-6 alpha proteinwill be expressed as a fusion protein (e.g., an Fc fusion protein or ahuman serum albumin fusion protein). The proteins expressed by theseexpression constructs are also encompassed by the present invention.

In other embodiments, the present invention provides TNFR-6 alphaexpression constructs that express fragments of TNFR-6 alpha and/orTNFR-6 beta containing the cysteine rich domains (e.g., amino acidresidues 1-195 of SEQ ID NO:2) either alone or as a fusion protein(e.g., an Fc fusion protein or a human serum albumin fusion protein).The proteins expressed by these expression constructs as well aspolynucleotides encoding the proteins expressed by these expressionconstructs, are also encompassed by the present invention.

In other embodiments, the present invention provides TNFR-6 alphaexpression constructs for expressing TNFR-6 alpha and/or TNFR-6 beta asa fusion protein with TR2 (SEQ ID NO:31, also described in InternationalPublication Numbers WO96/34095 and WO98/18824 which are hereinincorporated by reference in their entireties). In specific embodiments,the present invention encompasses an expression vector for expressing aTR2-TNFR-6 alphaTR2 fusion protein comprising amino acids M1-S41 of TR2(SEQ ID NO:31) fused to C48-S195 of TNFR-6 alpha (SEQ ID NO:2) fused toS186-A192 of TR2 (SEQ ID NO:31). In other embodiments the TR2-TNFR-6alpha-TR2 fusion protein is additionally fused to an immunoglobulin Fcregion or to human serum albumin. The proteins expressed by theseexpression constructs as well as polynucleotides encoding the proteinsexpressed by these expression constructs, are also encompassed by thepresent invention.

In other embodiments, the present invention provides TNFR-6 alphaexpression constructs for expressing TNFR-6 alpha fusions proteins inwhich the last 6 amino acids of TNFR-6 alpha have been deleted andreplaced with the amino acid sequence NIT. Such fusion proteins have theTNFR-6 alpha protein N-terminal of the fusion protein moiety (e.g., animmunoglobulin Fc region or human serum albumin). The NIT sequence mayserve as a glycosylation site. As a non-limiting mechanism, thecarbohydrate moieties on a glycosylated TNFR-6 alpha (M1-E294 of SEQ IDNO:2)-NIT-fusion protein may mask the fusion protein junction andprevent cleavage of the protein in the host cell. The TNFR-6 alpha(M1-E294 of SEQ ID NO:2)-NIT-fusion proteins (glycosylated andnon-glycosylated) are also encompassed by the present invention, as arepolynucleotides encoding the TNFR-6 alpha (M1-E294 of SEQ IDNO:2)-NIT-fusion proteins.

The present invention encompasses TNFR-6 alpha proteins which containalanine-160 to aspargine (A160N) and/or serine-186 to asparagine (S186N)point mutations. The present invention also encompasses TNFR-6 alpha(A160N, S186N) fusion polypeptides (e.g., TNFR6-alpha (A160N, S186N)fused to an immunoglobulin Fc domain or to human serum albumin).Polynucleotides encoding these TNFR-6 alpha (A160N, S186N) polypeptides(both fusion and non-fusion) as well as vectors comprisingpolynucleotides encoding these TNFR-6 alpha (A160N, S186N) polypeptides,are also encompassed by the invention.

In other embodiments, the present invention provides TNFR-6 alphaexpression constructs which comprise a polynucleotide encoding mammaliansynthetic TNFR-6 alpha (SEQ ID NO:32). In preferred embodiments, thepresent invention provides TNFR-6 alpha expression constructs whichcomprise a polynucleotide encoding mammalian synthetic TNFR-6 alpha (SEQID NO:32) operably linked to a heterologous regulatory sequence. Instill other embodiments, the present invention provides TNFR-6 alphaexpression constructs which comprise a polynucleotide encoding mammaliansynthetic TNFR-6 alpha (SEQ ID NO:32) fused in frame to a polynucleotideencoding a heterologous polypeptide, such as a polynucleotide encodingan immunoglobulin constant domain or human serum albumin.

In other embodiments, the present invention provides TNFR-6 alphaexpression constructs which comprise a polynucleotide encoding TNFR-6alpha which has been codon optimized for expression in yeast (SEQ IDNO:33). In preferred embodiments, the present invention provides TNFR-6alpha expression constructs which comprise polynucleotide encodingTNFR-6 alpha which has been codon optimized for expression in yeast (SEQID NO:33) operably linked to a heterologous regulatory sequence. Instill other embodiments, the present invention provides TNFR-6 alphaexpression constructs which polynucleotide encoding TNFR-6 alpha whichhas been codon optimized for expression in yeast (SEQ ID NO:33) fused inframe to a polynucleotide encoding a heterologous polypeptide such as, apolynucleotide encoding an immunoglobulin constant domain or human serumalbumin.

Proteins of the present invention include: products purified fromnatural sources, including bodily fluids, tissues and cells, whetherdirectly isolated or cultured; products of chemical syntheticprocedures; and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial,yeast, higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the polypeptides of thepresent invention may be glycosylated or may be non-glycosylated. Inaddition, polypeptides of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses.

Proteins of the invention can be chemically synthesized using techniquesknown in the art (e.g., see Creighton, 1983, Proteins: Structures andMolecular Principles, W. H. Freeman & Co., N.Y., and Hunkapiller, M., etal., Nature 310:105-111 (1984)). For example, a peptide corresponding toa fragment of the complete TNFR (i.e., TNFR-6α and/or TNFR-6β)polypeptides of the invention can be synthesized by use of a peptidesynthesizer. Furthermore, if desired, nonclassical amino acids orchemical amino acid analogs can be introduced as a substitution oraddition into the TNFR polypeptide sequence. Non-classical amino acidsinclude, but are not limited to, to the D-isomers of the common aminoacids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyricacid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid,Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine,norleucine, norvaline, hydroxyproline, sarcosine, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids,designer amino acids such as b-methyl amino acids, Ca-methyl aminoacids, Na-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The TNFR-6 alpha and/or TNFR-6 beta proteins may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given TNFR-6 alpha and/orTNFR-6 beta protein. Also, a given TNFR-6 alpha and/or TNFR-6 betaprotein may contain many types of modifications. TNFR-6 alpha and/orTNFR-6 beta proteins may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic TNFR-6 alpha and/or TNFR-6 betaproteins may result from posttranslation natural processes or may bemade by synthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993);POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., MethEnzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62(1992).)

The invention encompasses TNFR-6α and/or TNFR-6β proteins which aredifferentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited to, specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

The present invention further encompasses TNFR-6α and/orTNFR-6βpolypeptides or fragments thereof conjugated to a diagnosticagent (e.g. a detecable agent) and/or therapeutic agent. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to thepolypeptide (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to polypeptides for use as diagnostics and/ortherapeutics according to the present invention. Examples of suitableenzymes include horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include iodine (¹²¹I,¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium(¹¹¹In, ¹¹²In, ^(113m)In, ^(115m)In), technetium (⁹⁹Tc, ^(99m)Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³ Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru. Apreferred radioisotope label is ¹¹¹I. Another preferred radioactivelabel is ⁹⁰Y. Another preferred radioactive label is ¹³¹I.

Further, TNFR-6α and/or TNFR-6β polypeptides or fragments or variantsthereof may be conjugated to a therapeutic moiety such as a cytotoxin,e.g., a cytostatic or cytocidal agent, a therapeutic agent or aradioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bior other radioisotopes such as, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se,¹¹³Sn, ⁹⁰Y, ¹¹⁷Tin, ¹⁸⁶Re, ¹⁸⁸Re and ¹⁶⁶Ho. In specific embodiments,TNFR-6α and/or TNFR-6β polypeptides or fragments or variants thereof areattached to macrocyclic chelators useful for conjugating radiometalions, including but not limited to, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, topolypeptides. In a preferred embodiment, the radiometal ion associatedwith the macrocyclic chelators attached to TNFR-6α and/or TNFR-6polypeptides of the invention is ¹¹¹In. In another preferred embodiment,the radiometal ion associated with the macrocyclic chelator attached toTNFR-6α and/or TNFR-6β polypeptides of the invention is ⁹⁰Y. In specificembodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inother specific embodiments, the DOTA is attached to the an antibody ofthe invention or fragment thereof via a linker molecule. Examples oflinker molecules useful for conjugating DOTA to a polypeptide arecommonly known in the art—see, for example, DeNardo et al., Clin CancerRes. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7,1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999 which arehereby incorporated by reference in their entirety. In addition U.S.Pat. Nos. 5,652,361 and 5,756,065, which disclose chelating agents thatmay be conjugated to antibodies, and methods for making and using them,are hereby incorporated by reference in their entireties.

Techniques known in the art may be applied to label antibodies of theinvention. Such techniques include, but are not limited to, the use ofbifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065;5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contentsof each of which are hereby incorporated by reference in its entirety)and direct coupling reactions (e.g., Bolton-Hunter and Chloramine-Treaction).

Also provided by the invention are chemically modified derivatives ofTNFR-6α and/or TNFR-6β which may provide additional advantages such asincreased solubility, stability and circulating time of the polypeptide,or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemicalmoieties for derivitization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to a proteins via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the protein or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may beaccomplished by any number of means. For example, polyethylene glycolmay be attached to the protein either directly or by an interveninglinker. Linkerless systems for attaching polyethylene glycol to proteinsare described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998);U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO98/32466, the disclosures of each of which are incorporated herein byreference.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monmethoxy polyethyleneglycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction ofprotein with tresylated MPEG, polyethylene glycol is directly attachedto amine groups of the protein. Thus, the invention includesprotein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin WO 98/32466, the entire disclosure of which is incorporated herein byreference. Pegylated protein products produced using the reactionchemistries set out herein are included within the scope of theinvention.

The number of polyethylene glycol moieties attached to each protein ofthe invention (ie., the degree of substitution) may also vary. Forexample, the pegylated proteins of the invention may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

The TNFR proteins can be recovered and purified by known methods whichinclude, but are not limited to, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification.

TNFR Proteins

The invention further provides for the proteins containing polypeptidesequences encoded by the polynucleotides of the invention.

The TNFR proteins of the invention may be in monomers or multimers(i.e., dimers, trimers, tetramers, and higher multimers). Accordingly,the present invention relates to monomers and multimers of the TNFRproteins of the invention, their preparation, and compositions(preferably, pharmaceutical compositions) containing them. In specificembodiments, the polypeptides of the invention are monomers, dimers,trimers or tetramers. In additional embodiments, the multimers of theinvention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing only TNFRproteins of the invention (including TNFR fragments, variants, andfusion proteins, as described herein). These homomers may contain TNFRproteins having identical or different polypeptide sequences. In aspecific embodiment, a homomer of the invention is a multimer containingonly TNFR proteins having an identical polypeptide sequence. In anotherspecific embodiment, a homomer of the invention is a multimer containingTNFR proteins having different polypeptide sequences. In specificembodiments, the multimer of the invention is a homodimer (e.g.,containing TNFR proteins having identical or different polypeptidesequences) or a homotrimer (e.g., containing TNFR proteins havingidentical or different polypeptide sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containingheterologous proteins (i.e., proteins containing only polypeptidesequences that do not correspond to a polypeptide sequences encoded bythe TNFR gene) in addition to the TNFR proteins of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when proteins of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when proteins of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the TNFR proteins of the invention.Such covalent associations may involve one or more amino acid residuescontained in the polypeptide sequence of the protein ( e.g., thepolypeptide sequence recited in SEQ ID NO:2 or SEQ ID NO:4, contained inthe polypeptide encoded by the cDNA clone contained in ATCC Deposit No.97810), contained in the polypeptide encoded by the cDNA clone containedin ATCC Deposit No. 97809). In one instance, the covalent associationsare cross-linking between cysteine residues located within thepolypeptide sequences of the proteins which interact in the native(i.e., naturally occurring) polypeptide. In another instance, thecovalent associations are the consequence of chemical or recombinantmanipulation. Alternatively, such covalent associations may involve oneor more amino acid residues contained in the heterologous polypeptidesequence in a TNFR fusion protein. In one example, covalent associationsare between the heterologous sequence contained in a fusion protein ofthe invention (see, e.g., U.S. Pat. No. 5,478,925). In a specificexample, the covalent associations are between the heterologous sequencecontained in a TNFR-Fc fusion protein of the invention (as describedherein). In another specific example, covalent associations of fusionproteins of the invention are between heterologous polypeptide sequencesfrom another TNF family ligand/receptor member that is capable offorming covalently associated multimers, such as for example,oseteoprotegerin (see, e.g., International application publicationnumber WO 98/49305, the contents of which are herein incorporated byreference in its entirety). In another embodiment, two or more TR6-alphaand/or TR6-beta polypeptides of the invention are joined through peptidelinkers. Examples include those peptide linkers described in U.S. Pat.No. 5,073,627 (hereby incorporated by reference). Proteins comprisingmultiple TR6-alpha and/or TR6-beta polypeptides separated by peptidelinkers may be produced using conventional recombinant DNA technology.

Another method for preparing multimer TR6-alpha and/or TR6-betapolypeptides of the invention involves use of TR6-alpha and/or TR6-betapolypeptides fused to a leucine zipper or isoleucine zipper polypeptidesequence. Leucine zipper and isoleucine zipper domains are polypeptidesthat promote multimerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., Science 240:1759, (1988)), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble multimeric TR6-alpha and/or TR6-beta proteins arethose described in PCT application WO 94/10308, hereby incorporated byreference. Recombinant fusion proteins comprising a soluble TR6-alphaand/or TR6-beta polypeptide fused to a peptide that dimerizes ortrimerizes in solution are expressed in suitable host cells, and theresulting soluble multimeric TR6-alpha and/or TR6-beta is recovered fromthe culture supernatant using techniques known in the art.

Certain members of the TNF family of proteins are believed to exist intrimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al.,i Cell 73:431, 1993). Thus, trimeric TR6-alpha and/or TR6-beta may offerthe advantage of enhanced biological activity. Preferred leucine zippermoieties are those that preferentially form trimers. One example is aleucine zipper derived from lung surfactant protein D (SPD), asdescribed in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S.patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric TR6-alpha and/orTR6-beta.

In further preferred embodiments, TR6-alpha or TR6-beta polynucleotidesof the invention are fused to a polynucleotide encoding a FLAG®polypeptide (DYKDDDDK). Thus, a TR6-alpha-FLAG or a TR6-beta-FLAG fusionprotein is encompassed by the present invention. The FLAG® antigenicpolypeptide may be fused to a TR6-alpha or a TR6-beta polypeptide of theinvention at either or both the amino or the carboxy terminus. Inpreferred embodiments, a TR6-alpha-FLAG or a TR6-beta-FLAG fusionprotein is expressed from a pFLAG-CMV™-5a or a pFLAG-CMV™-1 expressionvector (available from Sigma, St. Louis, Mo., USA). See, Andersson, S.,et al., J. Biol. Chem. 264:8222-29 (1989); Thomsen, D. R., et al., Proc.Natl. Acad. Sci. USA, 81:659-63 (1984); and Kozak, M., Nature 308:241(1984) (each of which is hereby incorporated by reference). In furtherpreferred embodiments, a TR6-alpha-FLAG or a TR6-beta-FLAG fusionprotein is detectable by anti-FLAG® monoclonal antibodies (alsoavailable from Sigma).

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, proteins desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the polypeptidesequence of the proteins desired to be contained in the multimer (see,e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by referencein its entirety). Further, proteins of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide sequence of the protein and techniquesknown in the art may be applied to generate multimers containing one ormore of these modified proteins (see, e.g., U.S. Pat. No. 5,478,925,which is herein incorporated by reference in its entirety).Additionally, techniques known in the art may be applied to generateliposomes containing the protein components desired to be contained inthe multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, proteinscontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain and which can beincorporated by membrane reconstitution techniques into liposomes (see,e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by referencein its entirety).

In one embodiment, the invention provides isolated TNFR proteinscomprising, or alternatively, consisting of, the amino acid sequence ofthe complete (full-length) TNFR polypeptide encoded by the cDNAcontained in ATCC Deposit No. 97810, the amino acid sequence of thecomplete (full-length) TNFR polypeptide encoded by the cDNA contained inATCC Deposit No. 97809, the amino acid sequence of the complete TNFR-6αpolypeptide disclosed in FIG. 1 (SEQ ID NO:2), the amino acid sequenceof the complete TNFR-6β polypeptide disclosed in FIG. 2A (SEQ ID NO:4),or a portion of the above polypeptides.

In another embodiment, the invention provides isolated TNFR proteinscomprising, or alternatively consisting of, the amino acid sequence ofthe mature TNFR polypeptide encoded by the cDNA contained in ATCCDeposit No. 97810, the amino acid sequence of the mature TNFRpolypeptide encoded by the cDNA contained in ATCC Deposit No. 97809,amino acid residues 31 to 300 of the TNFR-6α sequence disclosed in FIG.1 (SEQ ID NO:2), amino acid residues 31 to 170 of the TNFR-6β sequencedisclosed in FIG. 2 (SEQ ID NO:4), or a portion (i.e., fragment) of theabove polypeptides.

Polypeptide fragments of the present invention include polypeptidescomprising or alternatively, consisting of, an amino acid sequencecontained in SEQ ID NO:2, an amino acid sequence contained in SEQ IDNO:4, an amino acid sequence encoded by the cDNA plasmid deposited asATCC Deposit No. 97810, an amino acid sequence encoded by the cDNAplasmid deposited as ATCC Deposit No. 97809, or an amino acid sequenceencoded by a nucleic acid which hybridizes (e.g., under stringenthybridization conditions) to the nucleotide sequence of the cDNAcontained in ATCC Deposit No. 97810 and/or 97809, or shown in FIGS. 1and/or 2 (SEQ ID NO:1 and SEQ ID NO:3, respectively) or thecomplementary strand thereto. Polynucleotides that hybridize to thesepolynucleotide fragments are also encompassed by the invention. Proteinfragments may be “free-standing,” or comprised within a largerpolypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentsthat comprise or alternatively, consist of from amino acid residues: Ito 31, 32 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to 250, and/or251 to 300 of SEQ ID NO:2. Additional representative examples ofpolypeptide fragments of the invention include polypeptide fragmentsthat comprise, or alternatively, consist of from amino acids 1 to 31, 32to 70, 70 to 100, 100 to 125, 126 to 150, and/or 151 to 170 of SEQ IDNO:4. Moreover, polypeptide fragments can be at least 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acidsin length. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

In specific embodiments, polypeptide fragments of the inventioncomprise, or alternatively consist of, amino acid residues: 100 to 150,150 to 200, 200 to 300, 210 to 300, 220 to 300, 230 to 300, 240 to 300,250 to 300, 260 to 300, 270 to 300, 280 to 300, and/or 290 to 300 asdepicted in FIG. 1 (SEQ ID NO:2). Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

TNFR comprises two domains having different structural and functionalproperties. The amino terminal domain spanning residues 30 to 196 of SEQID NO:2 shows homology to other members of the TNFR family, throughconservation of four cysteine rich domains characteristic of TNFRfamilies. Amino acid sequences contained in each of the four domainsinclude amino acid residues 34 to 70, 73 to 113, 115 to 150, and 153 to193, of SEQ ID NO:2, respectively. The carboxy terminal domain, spanningamino acid residues 197 to 300 of SEQ ID NO:2, has no significanthomology to any known sequences. Unlike a number of other TNF receptorfamily members, TNFR appears to be exclusively a secreted protein anddoes not appear to be synthesized as a membrane associated form. Whilethe amino terminal domain of TNFR appears to be required for biologicalactivity of TNFR, the carboxy-terminal domain appears to be importantfor multimerization of TNFR.

In one embodiment, the polypeptides of the invention comprise, oralternatively consist of, amino acid residues 34 to 70, 73 to 113, 115to 150, and 153 to 193, and/or 30-196 of SEQ ID NO:2. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

In another embodiment, the polypeptides of the invention comprise, oralternatively consist of, amino acid residues 197 to 240, 241 to 270,271-300, and/or 197 to 300 of SEQ ID NO:2. Polynucleotides encodingthese polypeptides are also encompassed by the invention. Since thesepolypeptide sequences are believed to be associated with multimerizationof TNFR, proteins having one or more of these polypeptide sequenceswould be expected to form dimers, trimers and higher multimers, whichmay have advantageous properties, such as, increased binding affinity,greater stability, and longer circulating half life compared tomonomeric forms. In a specific embodiment, the invention provides forfusion proteins comprising fusions of one or more of the abovepolypeptides to a heterologous sequence of a cell signaling molecule,such as a receptor, an extracellular domain thereof, and an activefragment, derivative, or analog of a receptor or an extracellulardomain. In a preferred embodiment, heterologous sequences are selectedfrom the family of TNR-like receptors. Such sequences preferably includefunctional extracellular ligand binding domains and lack functionaltransmembrane and/or cytoplasmic domains. Such fusion proteins areuseful for detecting molecules which interact with the fusedheterologous sequences and thereby identifying potential new receptorsand ligands. The fusion proteins are also useful for treatment of avariety of disorders, for example, those related to receptor binding. Inone embodiment, fusion proteins of the invention comprising TNF/TNFR andTNF receptor/TNFR sequences are used to treat TNF and TNF receptormediated disorders, such as, inflammation, autoimmune diseases, cancer,and disorders associated with excessive or alternatively, reducedapoptosis.

Additional embodiments TNFR polypeptide fragments comprising, oralternatively, consisting of, functional regions of polypeptides of theinvention, such as the Gamier-Robson alpha-regions, beta-regions,turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions,and coil-regions, Kyte-Doolittle hydrophilic regions, Eisenberg alpha-and beta-amphipathic regions, Karplus-Schulz flexible regions, Eminisurface-forming regions and Jameson-Wolf regions of high antigenic indexset out in FIG. 4 (Table I) and FIG. 5 (Table 2) and as describedherein. In a preferred embodiment, the polypeptide fragments of theinvention are antigenic. The data presented in columns VIII, IX, XIII,and XIV of Tables I and II can be used to routinely determine regions ofTNFR which exhibit a high degree of potential for antigenicity. Regionsof high antigenicity are determined from the data presented in columnsVIII, IX, XIII, and/or XIV by choosing values which represent regions ofthe polypeptide which are likely to be exposed on the surface of thepolypeptide in an environment in which antigen recognition may occur inthe process of initiation of an immune response. Among highly preferredfragments of the invention are those that comprise regions of TNFR thatcombine several structural features, such as several (e.g., 1, 2, 3 or4) of the features set out above. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NOS:2 and 4, respectively, or an epitopeof the polypeptide sequence encoded by a polynucleotide sequencecontained in deposited clone ATCC Deposit Number 97810 and 97809,respectively, or encoded by a polynucleotide that hybridizes to thecomplement of the sequence of SEQ ID NOS:1 and 3, respectively, orcontained in deposited clone ATCC Deposit Number 97810 and 97809,respectively, under stringent hybridization conditions or lowerstringency hybridization conditions as defined supra. The presentinvention further encompasses polynucleotide sequences encoding anepitope of a polypeptide sequence of the invention (such as, forexample, the sequence disclosed in SEQ ID NOS:1 and/or 3),polynucleotide sequences of the complementary strand of a polynucleotidesequence encoding an epitope of the invention, and polynucleotidesequences which hybridize to the complementary strand under stringenthybridization conditions or lower stringency hybridization conditionsdefined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate TNFR-specific antibodies include: a polypeptidecomprising, or alternatively consisting of, amino acid residues fromabout Ala-31 to about Thr-46, from about Phe-57 to about Thr-117, fromabout Cys-132 to about Thr-175, from about Gly-185 to about Thr-194,from about Val-205 to about Asp-217, from about Pro-239 to aboutLeu-264, and from about Ala-283 to about Pro-298 in SEQ ID NO:2; andfrom about Ala-31 to about Thr-46, from about Phe-57 to about Gln-80,from about Glu-86 to about His-106, from about Thr-108 to about Phe-119,from about His-129 to about Val-138, and from about Gly-142 to aboutPro-166 in SEQ ID NO:4. These polypeptide fragments have been determinedto bear antigenic epitopes of the TNFR-6 alpha and TNFR-6 betapolypeptides respectively, by the analysis of the Jameson-Wolf antigenicindex, as shown in FIGS. 4 and 5, above.

Fragments that function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 15, at least20, at least 25, and, most preferably, between about 15 to about 30amino acids. Preferred polypeptides comprising immunogenic or antigenicepitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenicepitopes are useful, for example, to raise antibodies, includingmonoclonal antibodies, that specifically bind the epitope. Antigenicepitopes can be used as the target molecules in immunoassays. (See, forinstance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al.,Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). The polypeptides comprising one or more immunogenicepitopes may be presented for eliciting an antibody response togetherwith a carrier protein, such as an albumin, to an animal system (suchas, for example, rabbit or mouse), or, if the polypeptide is ofsufficient length (at least about 25 amino acids), the polypeptide maybe presented without a carrier. However, immunogenic epitopes comprisingas few as 8 to 10 amino acids have been shown to be sufficient to raiseantibodies capable of binding to, at the very least, linear epitopes ina denatured polypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde.

Epitope bearing peptides of the invention may also be synthesized asmultiple antigen peptides (MAPs), first described by J. P. Tam in Proc.Natl. Acad Sci. USA. 85:5409 which is incorporated by reference hereinin its entirety. MAPs consist of multiple copies of a specific peptideattached to a non-immunogenic lysine core. Map peptides usually containfour or eight copies of the peptide often referred to as MAP-4 or MAP-8peptides. By way of non-limiting example, MAPs may be synthesized onto alysine core matrix attached to a polyethylene glycol-polystyrene(PEG-PS) support. The peptide of interest is synthesized onto the lysineresidues using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. For example,Applied Biosystems (Foster City, Calif.) offers MAP resins, such as, forexample, the Fmoc Resin 4 Branch and the Fmoc Resin 8 Branch which canbe used to synthesize MAPs. Cleavage of MAPs from the resin is performedwith standard trifloroacetic acid (TFA)-based cocktails known in theart. Purification of MAPs, except for desalting, is not necessary. MAPpeptides may be used as an immunizing vaccine which elicits antibodiesthat recognize both the MAP and the native protein from which thepeptide was derived.

Epitope bearing polypeptides of the invention may be modified, forexample, by the addition of amino acids at the amino- and/or carboxy-termini of the peptide. Such modifications may be performed, forexample, to alter the conformation of the epitope bearing polypeptidesuch that the epitope will have a conformation more closely related tothe structure of the epitope in the native protein. An example of amodified epitope-bearing polypeptide of the invention is a polypeptidein which one or more cysteine residues have been added to thepolypeptide to allow for the formation of a disulfide bond between twocysteines, resulting in a stable loop structure of the epitope bearingpolypeptide under non-reducing conditions. Disulfide bonds may formbetween a cysteine residue added to the polypeptide and a cysteineresidue of the naturally occurring epitope, or may form bewteen twocysteines which have both been added to the naturally ocurring epitopebearing polypeptide. Additionally, it is possible to modify one or moreamino acid residues of the naturally occurring epitope bearingpolypeptide by substituting them with cysteines to promote the formationof disulfide bonded loop structures. Cyclic thioether molecules ofsynthetic peptides may be routinely generated using techniques known inthe art and are described in PCT publication WO 97/46251, incorporatedin its entirety by reference herein. Other modifications ofepitope-bearing polypeptides contemplated by this invention includebiotinylation.

Animals such as, for example, rabbits, rats, and mice are immunized witheither free or carrier-coupled peptides, or MAP peptides, for instance,by intraperitoneal and/or intradermal injection of emulsions containingabout 100 micrograms of peptide or carrier protein and Freund's adjuvantor any other adjuvant known for stimulating an immune response. Severalbooster injections may be needed, for instance, at intervals of abouttwo weeks, to provide a useful titer of anti-peptide antibody that canbe detected, for example, by ELISA assay using free peptide adsorbed toa solid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention (e.g., those comprising animmunogenic or antigenic epitope) can be fused to heterologouspolypeptide sequences. For example, polypeptides of the presentinvention (including fragments or variants thereof), may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof,resulting in chimeric polypeptides. By way of another non-limitingexample, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) may be fused with albumin(including but not limited to recombinant human serum albumin orfragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, herein incorporated by reference in theirentirety)). In a preferred embodiment, polypeptides and/or antibodies ofthe present invention (including fragments or variants thereof) arefused with the mature form of human serum albumin (i.e., amino acids1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0322 094) which is herein incorporated by reference in its entirety. Inanother preferred embodiment, polypeptides and/or antibodies of thepresent invention (including fragments or variants thereof) are fusedwith polypeptide fragments comprising, or alternatively consisting of,amino acid residues 1-z of human serum albumin, where z is an integerfrom 369 to 419, as described in U.S. Pat. No. 5,766,883 hereinincorporated by reference in its entirety. Polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) may be fused to either the N— or C-terminal end of theheterologous protein (e.g., immunoglobulin Fc polypeptide or human serumalbumin polypeptide). Polynucleotides encoding fusion proteins of theinvention are also encompassed by the invention.

Such fusion proteins as those described above may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). IgG Fusion proteins thathave a disulfide-linked dimeric structure due to the IgG portiondesulfide bonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or FLAG® tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix-binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

The techniques of gene-shuffling, motif-shuffling, exon-shuffling,and/or codon-shuffling (collectively referred to as “DNA shuffling”) maybe employed to modulate the activities of TR6-alpha and/or TR6-betathereby effectively generating agonists and antagonists of TR6-alphaand/or TR6-beta. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238,5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr.Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol.16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76(1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13(1998) (each of these patents and publications are hereby incorporatedby reference). In one embodiment, alteration of TR6-alpha and/orTR6-beta polynucleotides and corresponding polypeptides may be achievedby DNA shuffling. DNA shuffling involves the assembly of two or more DNAsegments into a desired TR6-alpha and/or TR6-beta molecule byhomologous, or site-specific, recombination. In another embodiment,TR6-alpha and/or TR6-beta polynucleotides and corresponding polypeptidesmay be alterred by being subjected to random mutagenesis by error-pronePCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of TR6-alpha and/or TR6-betamay be recombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules. Inpreferred embodiments, the heterologous molecules are TNF-alpha,TNF-beta, lymphotoxin-alpha, lymphotoxin-beta, FAS ligand, APRIL. Infurther preferred embodiments, the heterologous molecules are any memberof the TNF family.

Additionally, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of TNFR therebyeffectively generating agonists and antagonists of TNFR. See generally,U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997);Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson et al., J. Mol.Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques24(2):308-13 (1998) (each of these patents and publications are herebyincorporated by reference). In one embodiment, alteration of TNFRpolynucleotides and corresponding polypeptides may be achieved by DNAshuffling. DNA shuffling involves the assembly of two or more DNAsegments into a desired TNFR molecule by homologous, or site-specific,recombination. In another embodiment, TNFR polynucleotides andcorresponding polypeptides may be altered by being subjected to randommutagenesis by error-prone PCR, random nucleotide insertion or othermethods prior to recombination. In another embodiment, one or morecomponents, motifs, sections, parts, domains, fragments, etc., of TNFRmay be recombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules. Inpreferred embodiments, the heterologous molecules include, but are notlimited to, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL,FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (InternationalPublication No. WO 96/14328), TRAIL, AIM-II (International PublicationNo. WO 97/34911), APRIL (J. Exp. Med 188(6):1185-1190), endokine-alpha(International Publication No. WO 98/07880), neutrokine alpha(International Publication No. WO98/18921), TR6 (InternationalPublication No. WO 98/30694), OPG, OX40, and nerve growth factor (NGF),and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (InternationalPublication No. WO 96/34095), DR3 (International Publication No. WO97/33904), DR4 (International Publication No. WO 98/32856), TR5(International Publication No. WO 98/30693), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2(International Publication No. WO 98/06842), and TR12, and soluble formsCD154, CD70, and CD153. In further preferred embodiments, theheterologous molecules are any member of the TNF family.

To improve or alter the characteristics of a TNFR polypeptide, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins or“muteins” including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions. For instance, for many proteins, including theextracellular domain of a membrane associated protein or the matureform(s) of a secreted protein, it is known in the art that one or moreamino acids may be deleted from the N-terminus or C-terminus withoutsubstantial loss of biological function. For instance, Ron et al., J.Biol. Chem., 268:2984-2988 (1993) reported modified KGF proteins thathad heparin binding activity even if 3, 8, or 27 amino-terminal aminoacid residues were missing.

In the present case, since the proteins of the invention are members ofthe TNFR polypeptide family, deletions of N-terminal amino acids up tothe Cysteine at position 49 of SEQ ID NOS:2 and 4 (TNFR-6 alpha andTNFR-6 beta) may retain some biological activity such as, for exampleregulation of cellular proliferation and apoptosis (e.g., of lymphoidcells), ability to bind Fas ligand (FasL), and ability to bind AIM-II.Polypeptides having further N-terminal deletions including the Cys-49residue in SEQ ID NOS:2 and 4, would not be expected to retain suchbiological activities because it is known that these residues in aTNFR-related polypeptide are required for forming a disulfide bridge toprovide structural stability which is needed for receptor/ligand bindingand signal transduction. However, even if deletion of one or more aminoacids from the N-terminus of a protein results in modification of lossof one or more biological functions of the protein, other functionalactivities may still be retained. Thus, the ability of the shortenedprotein to induce and/or bind to antibodies which recognize the completeor mature TNFR or extracellular domain of TNFR protein generally will beretained when less than the majority of the residues of the completeTNFR, mature TNFR, or extracellular domain of TNFR are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

Accordingly, the present invention further provides polypeptidescomprising or alternatively consisting of, one or more residues deletedfrom the amino terminus of the amino acid sequence of the TNFR shown inSEQ ID NOS:2 and 4, up to the cysteine residue at position number 49,and polynucleotides encoding such polypeptides. In particular, thepresent invention provides TNFR polypeptides comprising, oralternatively consisting of, the amino acid sequence of residues m-300of FIG. 1 (SEQ ID NO:2) and/or residues n-170 of FIG. 2A (SEQ ID NO:4),where m and n are integers in the range of 1-49 and where 49 is theposition of the first cysteine residue from the N-terminus of thecomplete TNFR-6α and TNFR-6β polypeptides (shown in SEQ ID NOS:2 and 4,respectively) believed to be required for activity of the TNFR-6α andTNFR-6β proteins.

More in particular, the invention provides polynucleotides encodingpolypeptides having (i.e., comprising) or alternatively consisting of,the amino acid sequence of a member selected from the group consistingof residues: 1-300, 2-300, 3-300, 4-300, 5-300, 6-300, 7-300, 8-300,9-300, 10-300, 11-300, 12-300, 13-300, 14-300, 15-300, 16-300, 17-300,18-300, 19-300, 20-300, 21-300, 22-300, 23-300, 24-300, 25-300, 26-300,27-300, 28-300, 29-300, 30-300, 31-300, 32-300, 33-300, 34-300, 35-300,36-300, 37-300, 38-300, 39-300, 40-300, 41-300, 42-300, 43-300, 44-300,45-300, 46-300, 47-300, 48-300, and 49-300 of SEQ ID NO:2; and 1-170,2-170, 3-170, 4-170, 5-170, 6-170, 7-170, 8-170, 9-170, 10-170, 11-170,12-170, 13-170, 14-170, 15-170, 16-170, 17-170, 18-170, 19-170, 20-170,21-170, 22-170, 23-170,24-170, 25-170, 26-170, 27-170, 28-170, 29-170,30-170, 31-170, 32-170, 33-170, 34-170, 35-170, 36-170, 37-170, 38-170,39-170, 40-170, 41-170, 42-170, 43-170, 44-170, 45-170, 46-170, 47-170,48-170, and 49-170 of SEQ ID NO:4. Polypeptides encoded by thesepolynucleotide fragments are also encompassed by the invention.

In a specific embodiment, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of a member selected from the group consisting ofresidues: Val-30 to His-300 of SEQ ID NO:2. Polypeptides encoded bythese polynucleotide fragments are also encompassed by the invention.

In other specific embodiments, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of a member selected from the group consisting ofresidues: P-23 to H-300, and/or P-34 to H-300 of SEQ ID NO:2.Polypeptides encoded by these polynucleotides are also encompassed bythe invention.

As mentioned above, even if deletion of one or more amino acids from theN-terminus of a protein results in modification of loss of one or morebiological functions of the protein, other functional activities (e.g.,biological activities) may still be retained. Thus, the ability ofshortened TNFR muteins to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptides generallywill be retained when less than the majority of the residues of thecomplete or mature polypeptide are removed from the N-terminus. Whethera particular polypeptide lacking N-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that a TNFR mutein with a large number ofdeleted N-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as six TNFRamino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the TNFR-6αamino acid sequence shown in FIG. 1 (i.e., SEQ ID NO:2), up to thearginine residue at position number 295 and polynucleotides encodingsuch polypeptides. In particular, the present invention providespolypeptides comprising or alternatively consisting of, the amino acidof residues n¹-300 of FIG. 1 (SEQ ID NO:2), where n¹ is an integer from49 to 295, corresponding to the position of the amino acid residue inFIG. 1 (SEQ ID NO:2).

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of a member selected from the group consisting of residues ofC49 to H-300; A-50 to H-300; Q-51 to H-300; C-52 to H-300; P-53 toH-300; P-54 to H-300; G-55 to H-300; T-56 to H-300; F-57 to H-300; V-58to H-300; Q-59 to H-300; R-60 to H-300; P-61 to H-300; C-62 to H-300;R-63 to H-300; R-64 to H-300; D-65 to H-300; S-66 to H-300; P-67 toH-300; T-68 to H-300; T-69 to H-300; C-70 to H-300; G-71 to H-300; P-72to H-300; C-73 to H-300; P-74 to H-300; P-75 to H-300; R-76 to H-300,H-77 to H-300; Y-78 to H-300; T-79 to H-300; Q-80 to H-300; F-81 toH-300; W-82 to H-300; N-83 to H-300; Y-84 to H-300; L-85 to H-300; E-86to H-300; R-87 to H-300; C-88 to H-300; R-89 to H-300; Y-90 to H-300;C-91 to H-300; N-92 to H-300; V-93 to H-300; L-94 to H-300; C-95 toH-300; G-96 to H-300; E-07 to H-300; R-98 to H-300; E-99 to H-300; E-100to H-300; E-101 to H-300; A-102 to H-300; R-103 to H-300; A-104 toH-300; C-105 to H-300; H-106 to H-300; A-107 to H-300; T-108 to H-300;H-109 to H-300; N-110 to H-300; R-111 to H-300; A-112 to H-300; C-113 toH-300; R-114 to H-300; C-115 to H-300; R-116 to H-300; T-117 to H-300;G-118 to H-300; F-119 to H-300; F-120 to H-300; A-121 to H-300; H-122 toH-300; A-123 to H-300; G-124 to H-300; F-125 to H-300; C-126 to H-300;L-127 to H-300; E-128 to H-300; H-129 to H-300; A-130 to H-300; S-131 toH-300; C-132 to H-300; P-133 to H-300; P-134 to H-300; G-135 to H-300;A-136 to H-300; G-137 to H-300; V-138 to H-300; I-139 to H-300; A-140 toH-300; P-141 to H-300; G-142 to H-300; T-143 to H-300; P-144 to H-300;S-145 to H-300; Q-146 to H-300; N-147 to H-300; T-148 to H-300; Q-149 toH-300; C-150 to H-300; Q-151 to H-300; P-152 to H-300; C-153 to H-300;P-154 to H-300; P-155 to H-300; G-156 to H-300; T-157 to H-300; F-158 toH-300; S-159 to H-300; A-160 to H-300; S-161 to H-300; S-162 to H-300;S-163 to H-300; S-164 to H-300; S-165 to H-300; E-166 to H-300; Q-167 toH-300; C-168 to H-300; Q-169 to H-300; P-170 to H-300; H-171 to H-300;R-172 to H-300; N-173 to H-300; C-174 to H-300; T-175 to H-300; A-176 toH-300; L-177 to H-300; G-178 to H-300; L-179 to H-300; A-180 to H-300;L-181 to H-300; N-182 to H-300; V-183 to H-300; P-184 to H-300; G-185 toH-300; S-186 to H-300; S-187 to H-300; S-188 to H-300; H-189 to H-300;D-190 to H-300; T-191 to H-300; L-192 to H-300; C-193 to H-300; T-194 toH-300; S-195 to H-300; C-196 to H-300; T-197 to H-300; G-198 to H-300;F-199 to H-300; P-200 to H-300; L-201 to H-300; S-202 to H-300; T-203 toH-300; R-204 to H-300; V-205 to H-300; P-206 to H-300; G-207 to H-300;A-208 to H-300; E-209 to H-300; E-210 to H-300; C-211 to H-300; E-212 toH-300; R-213 to H-300; A-214 to H-300; V-215 to H-300; I-216 to H-300;D-217 to H-300; F-218 to H-300; V-219 to H-300; A-220 to H-300; F-221 toH-300; Q-222 to H-300; D-223 to H-300; I-224 to H-300; S-225 to H-300;I-226 to H-300; K-227 to H-300; R-228 to H-300; L-229 to H-300; Q-230 toH-300; R-231 to H-300; L-232 to H-300; L-233 to H-300; Q-234 to H-300;A-235 to H-300; L-236 to H-300; E-237 to H-300; A-238 to H-300; P-239 toH-300; E-240 to H-300; G-241 to H-300; W-242 to H-300: G-243 to H-300;P-244 to H-300; T-245 to H-300; P-246 to H-300; R-247 to H-300; A-248 toH-300: G-249 to H-300; R-250 to H-300; A-251 to H-300; A-252 to H-300;L-253 to H-300; Q-254 to H-300; L-255 to H-300; K-256 to H-300; L-257 toH-300; R-258 to H-300; R-259 to H-300; R-260 to H-300; L-261 to H-300;T-262 to H-300; E-263 to H-300; L-264 to H-300; L-265 to H-300; G-266 toH-300; A-267 to H-300; Q-268 to H-300; D-269 to H-300; G-270 to H-300;A-271 to H-300; L-272 to H-300; L-273 to H-300; V-274 to H-300; R-275 toH-300; L-276 to H-300; L-277 to H-300; Q-278 to H-300; A-279 to H-300;L-280 to H-300; R-281 to H-300; V-282 to H-300; A-283 to H-300; R-284 toH-300; M-285 to H-300; P-286 to H-300; G-287 to H-300; L-288 to H-300;E-289 to H-300; R-290 to H-300; S-291 to H-300; V-292 to H-300; R-293 toH-300; E-294 to H-300; and R-295 to H-300 of the TNFR-6α sequence shownin FIG. 1 (SEQ ID NO:2). Polypeptides encoded by these polynucleotidefragments are also encompassed by the invention.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. For instance, interferon gamma shows up to ten timeshigher activities by deleting 8-10 amino acid residues from the carboxyterminus of the protein (Döbeli et al., J. Biotechnology 7:199-216(1988)). In the present case, since the protein of the invention is amember of the TNFR polypeptide family, deletions of C-terminal aminoacids up to the cysteine at position 193 and 132 of SEQ ID NOS:2 and 4,respectively, may retain some functional activity, such as, for example,a biological activity (such as, for example, regulation of proliferationand apoptosis (e.g., of lymphoid cells, ability to bind Fas ligand, andability to bind AIM-II)). Polypeptides having further C-terminaldeletions including the cysteines at positions 193 and 132 of SEQ IDNOS:2 and 4, respectively, would not be expected to retain suchbiological activities because it is known that these residues in TNFreceptor-related polypeptides are required for forming disulfide bridgesto provide structural stability which is needed for receptor binding.

However, even if deletion of one or more amino acids from the C-terminusof a protein results in modification or loss of one or more biologicalfunctions of the protein, other functional activities (e.g., biologicalactivities, the ability to multimerize, and the ability to bind ligand(e.g., Fas ligand and AIM-II)) may still be retained. Thus, the abilityof the shortened protein to induce and/or bind to antibodies whichrecognize the complete or mature form of the protein generally will beretained when less than the majority of the residues of the complete ormature form protein are removed from the C-terminus. Whether aparticular polypeptide lacking C-terminal residues of a complete proteinretains such immunologic activities can readily be determined by routinemethods described herein and otherwise known in the art.

Accordingly, the present invention further provides polypeptides havingone or more residues from the carboxy terminus of the amino acidsequence of TNFR-6 alpha and TNFR-6 beta shown in SEQ ID NOS:2 and 4 upto the cysteine at position 193 and 132 of SEQ ID NOS:2 and 4,respectively, and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising, oralternatively consisting of, the amino acid sequence of a memberselected from the group consisting of residues 1-y and 1-z of the aminoacid sequence in SEQ ID NOS:2 and 4, respectively, where y is anyinteger in the range of 193-300 and z is any integer in the range of132-170. Polynucleotides encoding these polypeptides also are provided.

In certain preferred embodiments, the present invention providespolypeptides comprising, or alternatively, consisting of, the amino acidsequence of a member selected from the group consisting of residues 1-y′and 1-z′ of the amino acid sequence in SEQ ID NOS:2 and 4, respectively,where y′ is any integer in the range of 193-299 and z′ is any integer inthe range of 132-169. Polynucleotides encoding these polypeptides alsoare provided.

In additional preferred specific embodiments, the present inventionprovides polypeptides comprising, or alternatively consisting of, theamino acid sequence of a member selected from the group consisting ofresidues Pro-23 to His-300, Val-30 to His-300, and Pro-34 to His-300 ofSEQ ID NO:2 and polypeptides having the amino acid sequence of a memberselected from the group consisting of residues Pro-23 to Pro-170, Val-30to Pro-170, and Pro-34 to His-Pro-170 of SEQ ID NO:4. As describedherein, these polypeptides may be fused to heterologous polypeptidesequences. Polynucleotides encoding these polypeptides and these fusionpolypeptides are also provided.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues m-y of SEQ ID NO:2 and n-z of SEQID NO:4, where m, n, y and z are integers as described above.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, the ability to form homomultimers, and theability to bind ligand (e.g., Fas ligand and AIM-II)) may still beretained. For example, the ability of the shortened TNFR mutein toinduce and/or bind to antibodies which recognize the complete or matureforms of the polypeptide generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the C-terminus. Whether a particular polypeptide lackingC-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a TNFR muteinwith a large number of deleted C-terminal amino acid residues may retainsome biological or immunogenic activities. In fact, peptides composed ofas few as six TNFR amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the TNFR polypeptide shown in FIG. 1 (SEQ ID NO:2), up tothe glycine residue at position number 6, and polynucleotides encodingsuch polypeptides. In particular, the present invention providespolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues 1-m¹ of FIG. 1 (i.e., SEQ ID NO:2), where m¹ is aninteger from 6 to 299, corresponding to the position of the amino acidresidue in FIG. 1 (SEQ ID NO:2).

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of a member selected from the group consisting of residues M-1to V-299; M-1 to P-298; M-1 to L-297; M-1 to F-296; M-1 to R-295; M-1 toE-294; M-1 to R-293; M-1 to V-292; M-1 to S-291; M-1 to R-290; M-1 toE-289; M-1 to L-288; M-1 to G-287; M-1 to P-286; M-1 to M-285; M-1 toR-284; M-1 to A-283; M-1 to V-282; M-1 to R-281; M-1 to L-280; M-1 toA-279; M-1 to Q-278; M-1 to L-277; M-1 to L-276; M-1 to R-275; M-1 toV-274; M-1 to L-273; M-1 to L-272; M-1 to A-271; M-1 to G-270; M-1 toD-269; M-1 to Q-268; M-1 to A-267; M-1 to G-266; M-1 to L-265; M-1 toL-264; M-1 to E-263; M-1 to T-262; M-1 to L-261; M-1 to R-260; M-1 toR-259; M-1 to R-258; M-1 to L-257; M-1 to K-256; M-1 to L-255; M-1 toQ-254; M-1 to L-253; M-1 to A-252; M-1 to A-251; M-1 to R-250; M-1 toG-249; M-1 to A-248; M-1 to R-247; M-1 to P-246; M-1 to T-245; M-1 toP-244; M-1 to G-243; M-1 to W-242; M-1 to G-241; M-1 to E-240; M-1 toP-239; M-1 to A-238; M-1 to E-237; M-1 to L-236; M-1 to A-235; M-1 toQ-234; M-1 to L-233; M-1 to L-232; M-1 to R-231; M-1 to Q-230; M-1 toL-229; M-1 to R-228; M-1 to K-227; M-1 to I-226; M-1 to S-225; M-1 toI-224; M-1 to D-223; M-1 to Q-222; M-1 to F-221; M-1 to A-220; M-1 toV-219; M-1 to F-218; M-1 to D-217; M-1 to I-216; M-1 to V-215; M-1 toA-214; M-1 to R-213; M-1 to E-212; M-1 to C-211; M-1 to E-210; M-1 toE-209; M-1 to A-208; M-1 to G-207; M-1 to P-206; M-1 to V-205; M-1 toR-204; M-1 to T-203; M-1 to S-202; M-1 to L-201; M-1 to P-200; M-1 toF-199; M-1 to G-198; M-1 to T-197; M-1 to C-196; M-1 to S-195; M-1 toT-194; M-1 to C-193; M-1 to L-192; M-1 to T-191; M-1 to D-190; M-1 toH-189; M-1 to S-188; M-1 to S-187; M-1 to S-186; M-1 to G-185; M-1 toP-184; M-1 to V-183; M-1 to N-182; M-1 to L-181; M-1 to A-180; M-1 toL-179; M-1 to G-178; M-1 to L-177; M-1 to A-176; M-1 to T-175; M-1 toC-174; M-1 to N-173; M-1 to R-172; M-1 to H-171; M-1 to P-170; M-1 toQ-169; M-1 to C-168; M-1 to Q-167; M-1 to E-166; M-1 to S-165; M-1 toS-164; M-1 to S-163; M-1 to S-162; M-1to S-161; M-1 to A-160; M-1 toS-159; M-1 to F-158; M-1 to T-157; M-1 to G-156; M-1 to P-155; M-1 toP-154; M-1 to C-153; M-1 to P-152; M-1 to Q-151; M-1 to C-150; M-1 toQ-149; M-1 to T-148; M-1 to N-147; M-1 to Q-146; M-1 to S-145; M-1 toP-144; M-1 to T-143; M-1 to G-142; M-1 to P-141; M-1 to A-140; M-1 toI-139; M-1 to V-138; M-1 to G-137; M-1 to A-136; M-1 to G-135; M-1 toP-134; M-1 to P-133; M-1 to C-132; M-1 to S-131; M-1 to A-130; M-1 toH-129; M-1 to E-128; M-1 to L-127; M-1 to C-126; M-1 to F-125; M-1 toG-124; M-1 to A-123; M-1 to H-122; M-1 to A-121; M-1 to F-120; M-1 toF-119; M-1 to G-118; M-1 to T-117; M-1 to R-116; M-1 to C-115; M-1 toR-114; M-1 to C-113; M-1 to A-112; M-1 to R-111; M-1 to N-110; M-1 toH-109; M-1 to T-108; M-1 to A-107; M-1 to H-106; M-1 to C-105; M-1 toA-104; M-1 to R-103; M-1 to A-102; M-1 to E-101; M-1 to E-100; M-1 toE-99; M-1 to R-98; M-1 to E-97; M-1 to G-96; M-1 to C-95; M-1 to L-94;M-1 to V-93; M-1 to N-92; M-1 to C-91; M-1 to Y-90; M-1 to R-89; M-1 toC-88; M-1 to R-87; M-1 to E-86; M-1 to L-85; M-1 to Y-84; M-1 to N-83;M-1 to W-82; M-1 to F-81; M-1 to Q-80; M-1 to T-79; M-1 to Y-78; M-1 toH-77; M-1 to R-76; M-1 to P-75; M-1 to P-74; M-1 to C-73; M-1 to P-72;M-1 to G-71; M-1 to C-70; M-1 to T-69; M-1 to T-68; M-1 to P-67; M-1 toS-66; M-1 to D-65; M-1 to R-64; M-1 to R-63; M-1 to C-62; M-1 to P-61;M-1 to R-60; M-1 to Q-59; M-1 to V-58; M-1 to F-57; M-1 to T-56; M-1 toG-55; M-1 to P-54; M-1 to P-53; M-1 to C-52; M-1 to Q-51; M-1 to A-50;M-1 to C-49; M-1 to V-48; M-1 to L-47; M-1 to R-46; M-1 to E-45; M-1 toG-44; M-1 to T-43; M-1 to E-42; M-1 to A41; M-1 to D-40; M-1 to R-39;M-1 to W-38; M-1 to P-37; M-1 to Y-36; M-1 to T-35; M-1 to P-34; M-1 toT-33; M-1 to E-32; M-1 to A-31; M-1 to V-30; M-1 to G-29; M-1 to R-28;M-1 to V-27; M-1 to A-26; M-1 to P-25; M-1 to V-24; M-1 to P-23; M-1 toL-22; M-1 to L-21; M-1 to A-20; M-1 to P-19; M-1 to L-18; M-1 to A-17;M-1 to L-16; M-1 to V-15; M-1 to L-14; M-1 to C-13; M-1 to L-12; M-1 toL-11; M-1 to S-10; M-1 to L-9; M-1 to G-8; M-1 to P-7; and M-1 to G-6 ofthe sequence of the TFNR sequence shown in FIG. 1 (SEQ ID NO:2).Polypeptides encoded by these polynucleotide fragments are alsoencompassed by the invention.

In specific embodiments, the invention provides polynucleotides encodingpolypeptides comprising or alternatively consisting of the amino acidsequence of a member selected from the group consisting of residues: M-1to A-271, M-1 to Q-254 and/or M-1 to F-221 of SEQ ID NO:2. Polypeptidesencoded by these polynucleotide fragments are also encompassed by theinvention.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini of a TNFRpolypeptide, which may be described generally as having residues n¹-m¹of FIG. 1 (i.e., SEQ ID NO:2), where n¹ and m¹ are integers as describedabove.

In additional embodiments, the present invention provides polypeptidescomprising or alternatively consisting of, the amino acid sequence ofresidues 30-m³ of FIG. 1 (i.e., SEQ ID NO:2), where m³ is an integerfrom 36 to 299, corresponding to the position of the amino acid residuein FIG. 1 (SEQ ID NO:2). For example, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of a member selected from thegroup consisting of residues V-30 to V-299; V-30 to P-298; V-30 toL-297; V-30 to F-296; V-30 to R-295; V-30 to E-294; V-30 to R-293; V-30to V-292; V-30 to S-291; V-30 to R-290; V-30 to E-289; V-30 to L-288;V-30 to G-287; V-30 to P-286; V-30 to M-285; V-30 to R-284; V-30 toA-283; V-30 to V-282; V-30 to R-281; V-30 to L-280; V-30 to A-279; V-30to Q-278; V-30 to L-277; V-30 to L-276; V-30 to R-275; V-30 to V-274;V-30 to L-273; V-30 to L-272; V-30 to A-271; V-30 to G-270; V-30 toD-269; V-30 to Q-268; V-30 to A-267; V-30 to G-266; V-30 to L-265; V-30to L-264; V-30 to E-263; V-30 to T-262; V-30 to L-261; V-30 to R-260;V-30 to R-259; V-30 to R-258; V-30 to L-257; V-30 to K-256; V-30 toL-255; V-30 to Q-254; V-30 to L-253; V-30 to A-252; V-30 to A-251; V-30to R-250; V-30 to G-249; V-30 to A-248; V-30 to R-247; V-30 to P-246;V-30 to T-245; V-30 to P-244; V-30 to G-243; V-30 to W-242; V-30 toG-241; V-30 to E-240; V-30 to P-239; V-30 to A-238; V-30 to E-237; V-30to L-236; V-30 to A-235; V-30 to Q-234; V-30 to L-233; V-30 to L-232;V-30 to R-231; V-30 to Q-230; V-30 to L-229; V-30 to R-228; V-30 toK-227; V-30 to I-226; V-30 to S-225; V-30 to I-224; V-30 to D-223; V-30to Q-222; V-30 to F-221; V-30 to A-220; V-30 to V-219; V-30 to F-218;V-30 to D-217; V-30 to I-216; V-30 to V-215; V-30 to A-214; V-30 toR-213; V-30 to E-212; V-30 to C-211; V-30 to E-210; V-30 to E-209; V-30to A-208; V-30 to G-207; V-30 to P-206; V-30 to V-205; V-30 to R-204;V-30 to T-203; V-30 to S-202; V-30 to L-201; V-30 to P-200; V-30 toF-199; V-30 to G-198; V-30 to T-197; V-30 to C-196; V-30 to S-195; V-30to T-194; V-30 to C-193; V-30 to L-192; V-30 to T-191; V-30 to D-190;V-30 to H-189; V-30 to S-188; V-30 to S-187; V-30 to S-186; V-30 toG-185; V-30 to P-184; V-30 to V-183; V-30 to N-182; V-30 to L-181; V-30to A-180; V-30 to L-179; V-30 to G-178; V-30 to L-177; V-30 to A-176;V-30 to T-175; V-30 to C-174; V-30 to N-173; V-30 to R-172; V-30 toH-171; V-30 to P-170; V-30 to Q-169; V-30 to C-168; V-30 to Q-167; V-30to E-166; V-30 to S-165; V-30 to S-164; V-30 to S-163; V-30 to S-162;V-30 to S-161; V-30 to A-160; V-30 to S-159; V-30 to F-158; V-30 toT-157; V-30 to G-156; V-30 to P-155; V-30 to P-154; V-30 to C-153; V-30to P-152; V-30 to Q-151; V-30 to C-150; V-30 to Q-149; V-30 to T-148;V-30 to N-147; V-30 to Q-146; V-30 to S-145; V-30 to P-144; V-30 toT-143; V-30 to G-142; V-30 to P-141; V-30 to A-140; V-30 to I-139; V-30to V-138; V-30 to G-137; V-30 to A-136; V-30 to G-135; V-30 to P-134;V-30 to P-133; V-30 to C-132; V-30 to S-131; V-30 to A-130; V-30 toH-129; V-30 to E-128; V-30 to L-127; V-30 to C-126; V-30 to F-125; V-30to G-124; V-30 to A-123; V-30 to H-122; V-30 to A-121; V-30 to F-120;V-30 to F-119; V-30 to G-118; V-30 to T-117; V-30 to R-116; V-30 toC-115; V-30 to R-114; V-30 to C-113; V-30 to A-112; V-30 to R-111; V-30to N-110; V-30 to H-109; V-30 to T-108; V-30 to A-107; V-30 to H-106;V-30 to C-105; V-30 to A-104; V-30 to R-103; V-30 to A-102; V-30 toE-101; V-30 to E-100; V-30 to E-99; V-30 to R-98; V-30 to E-97; V-30 toG-96; V-30 to C-95; V-30 to L-94; V-30 to V-93; V-30 to N-92; V-30 toC-91; V-30 to Y-90; V-30 to R-89; V-30 to C-88; V-30 to R-87; V-30 toE-86; V-30 to L-85; V-30 to Y-84; V-30 to N-83; V-30 to W-82; V-30 toF-81; V-30 to Q-80; V-30 to T-79; v-30 to Y-78; V-30 to H-77; V-30 toR-76; V-30 to P-75; V-30 to P-74; V-30 to C-73; V-30 to P-72; V-30 toG-71; V-30 to C-70; V-30 to T-69; V-30 to T-68; V-30 to P-67; V-30 toS-66; V-30 to D-65; V-30 to R-64; V-30 to R-63; V-30 to C-62; V-30 toP-61; V-30 to R-60; V-30 to Q-59; V-30 to V-58; V-30 to F-57; V-30 toT-56; V-30 to G-55; V-30 to P-54; V-30 to P-53; V-30 to C-52; V-30 toQ-51; V-30 to A-50; V-30 to C-49; V-30 to V-48; V-30 to L-47; V-30 toR-46; V-30 to E-45; V-30 to G-44; V-30 to T-43; V-30 to E-42; V-30 toA-41; V-30 to D-40; V-30 to R-39; V-30 to W-38; V-30 to P-37; and V-30to Y-36 of the sequence of the TFNR sequence shown in FIG. 1 (SEQ IDNO:2). Polypeptides encoded by these polynucleotide fragments are alsoencompassed by the invention. In specific embodiments, the inventionprovides polynucleotides encoding polypeptides comprising oralternatively consisting of the amino acid sequence of a member selectedfrom the group consisting of residues: V-30 to A-271, V-30 to Q-254and/or V-30 to F-221 of SEQ ID NO:2. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention. The presentapplication is also directed to polynucleotides or polypeptidescomprising, or alternatively, consisting of, a polynucleotide orpolypeptide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or99% identical to a polypeptide or polypeptide sequence described above,respectively. The present invention also encompasses the abovepolynucleotide or polypeptide sequences fused to a heterologouspolynucleotide or polypeptide sequence, respectively.

With respect to fragments of TNFR-6β, as mentioned above, even ifdeletion of one or more amino acids from the N-terminus of a proteinresults in modification of loss of one or more biological functions ofthe protein, other functional activities (e.g., biological activities,the ability to multimerize, the ability to bind ligand (e.g., Fas ligandand/or AIM-II)) may still be retained. For example, the ability ofshortened TNFR muteins to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptides generallywill be retained when less than the majority of the residues of thecomplete or mature polypeptide are removed from the N-terminus. Whethera particular polypeptide lacking N-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that a TNFR mutein with a large number ofdeleted N-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as six TNFRamino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptidescomprising, or alternatively, consisting of, one or more residuesdeleted from the amino terminus of the TNFR-6β amino acid sequence shownin FIG. 2A (i.e., SEQ ID NO:4), up to the glycine residue at positionnumber 165 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising, oralternatively consisting of, the amino acid sequence of residues n²-170of FIG. 2A (SEQ ID NO:4), where n² is an integer from 2 to 165,corresponding to the position of the amino acid residue in FIG. 2A (SEQID NO:4).

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of a member selected from the group consisting of residues ofR-2 to P-170; A-3 to P-170; L-4 to P-170; E-5 to P-170; G-6 to P-170;P-7 to P-170; L-9 to P-170; S-10 to P-170; L-11 to P-170; L-12 to P-170;C-13 to P-170; L-14 to P-170; V-15 to P-170; L-16 to P-170; A-17 toP-170; L-18 to P-170; P-19 to P-170; A-20 to P-170; L-21 to P-170; L-22to P-170; P-23 to P-170; V-24 to P-170; P-25 to P-170; A-26 to P-170;V-27 to P-170; R-28 to P-170; G-29 to P-170; V-30 to P-170; A-31 toP-170; E-32 to P-170; T-33 to P-170; P-34 to P-170; T-35 to P-170; Y-36to P-170; P-37 to P-170; W-38 to P-170; R-39 to P-170; D-40 to P-170;A-41 to P-170; E-42 to P-170; T-43 to P-170; G-44 to P-170; E-45 toP-170; R-46 to P-170; L-47 to P-170; V-48 to P-170; C-49 to P-170; A-50to P-170; Q-51 to P-170; C-52 to P-170; P-53 to P-170; P-54 to P-170;G-55 to P-170; T-56 to P-170; F-57 to P-170; V-58 to P-170; Q-59 toP-170; R-60 to P-170; P-61 to P-170; C-62 to P-170; R-63 to P-170; R-64to P-170; D-65 to P-170; S-66 to P-170; P-67 to P-170; T-68 to P-170;T-69 to P-170; C-70 to P-170; G-71 to P-170; P-72 to P-170; C-73 toP-170; P-74 to P-170; P-75 to P-170; R-76 to P-170; H-77 to P-170; Y-78to P-170; T-79 to P-170; Q-80 to P-170; F-81 to P-170; W-82 to P-170;N-83 to P-170; Y-84 to P-170; L-85 to P-170; E-86 to P-170; R-87 toP-170; C-88 to P-170; R-89 to P-170; Y-90 to p-170; C-91 to P-170; N-92to P-170; V-93 to P-170; L-94 to P-170; C-95 to P-170; G-96 to P-170;E-97 to P-170; R-98 to P-170; E-99 to P-170; E-100 to P-170; E-101 toP-170; A-102 to P-170; R-103 to P-170; A-104 to P-170; C-105 to P-170;H-106 to P-170; A-107 to P-170; T-108 to P-170; H-109 to P-170; N-110 toP-170; R-111 to P-170; A-112 to P-170; C-113 to P-170; R-114 to P-170;C-115 to P-170; R-116 to P-170; T-117 to P-170; G-118 to P-170; F-119 toP-170; F-120 to P-170; A-121 to P-170; H-122 to P-170; A-123 to P-170;G-124 to P-170; F-125 to P-170; C-126 to P-170; L-127 to P-170; E-128 toP-170; H-129 to P-170; A-130 to P-170; S-131 to P-170; C-132 to p-170;R-133 to P-170; P-134 to P-170; G-135 to P-170; A-136 to P-170; G-137 toP-170; V-138 to P-170; I-139 to P-170; A-140 to P-170; P-141 to P-170;G-142 to P-170; E-143 to P-170; S-144 to P-170; W-145 to P-170; A-146 toP-170; R-147 to P-170; G-148 to P-170; G-149 to P-170; A-150 to P-170;P-151 to P-170; R-152 to P-170; S-153 to P-170; G-154 to P-170; G-155 toP-170; R-156 to P-170; R-157 to P-170; C-158 to P-170; G-159 to P-170;R-160 to P-170; G-161 to P-170; Q-162 to P-170; V-163 to P-170; A-164 toP-170; and G-165 to P-170 of the TNFR-6β sequence shown in FIG. 2A (SEQID NO:4). Polypeptides encoded by these polynucleotide fragments arealso encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, the ability to multimerize, ability tobind ligand (e.g., Fas ligand and/or AIM-II) may still be retained. Forexample, the ability of the shortened TNFR-6β mutein to induce and/orbind to antibodies which recognize the complete or mature forms of thepolypeptide generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theC-terminus. Whether a particular polypeptide lacking C-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a TNFR-6β mutein with a largenumber of deleted C-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six TNFR-6β amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptidescomprising, or alternatively consisting of one or more residues deletedfrom the carboxy terminus of the amino acid sequence of the TNFR-6βpolypeptide shown in FIG. 2A (SEQ ID NO:4), up to the glycine residue atposition number 6, and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising, oralternatively consisting of, the amino acid sequence of residues 1-m² ofFIG. 2A (i.e., SEQ ID NO:4), where m² is an integer from 6 to 169,corresponding to the position of the amino acid residue in FIG. 2A (SEQID NO:4).

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of a member selected from the group consisting of residues M-1to A-169; M-1 to L-168; M-1 to S-167; M-1 to P-166; M-1 to G-165; M-1 toA-164; M-1 to V-163; M-1 to Q-162; M-1 to G-161; M-1 to R-160; M-1 toG-159; M-1 to C-158; M-1 to R-157; M-1 to R-156; M-1 to G-155; M-1 toG-154; M-1 to S-153; M-1 to R-152; M-1 to P-151; M-1 to A-150; M-1 toG-149; M-1 to G-148; M-1 to R-147; M-1 to A-146; M-1 to W-145; M-1 toS-144; M-1 to E-143; M-1 to G-142; M-1 to P-141; M-1 to A-140; M-1 toI-139; M-1 to V-138; M-1 to G-137; M-1 to A-136; M-1 to G-135; M-1 toP-134; M-1 to P-133; M-1 to C-132; M-1 to S-131; M-1 to A-130; M-1 toH-129; M-1 to E-128; M-1 to L-127; M-1 to C-126; M-1 to F-125; M-1 toG-124; M-1 to A-123; M-1 to H-122; M-1 to A-121; M-1 to F-120; M-1 toF-119; M-1 to G-118; M-1 to T-117; M-1 to R-116; M-1 to C-115; M-1 toR-114; M-1 to C-113; M-1 to A-112; M-1 to R-111; M-1 to N-110; M-1 toH-109; M-1 to T-108; M-1 to A-107; M-1 to H-106; M-1 to C-105; M-1 toA-104; M-1 to R-103; M-1 to A-102; M-1 to E-101; M-1 to E-100; M-1 toE-99; M-1 to R-98; M-1 to E-97; M-1 to G-96; M-1 to C-95; M-1 to L-94;M-1 to V-93; M-1 to N-92; M-1 to C-91; M-1 to Y-90; M-1 to R-89; M-1 toC-88; M-1 to R-87; M-1 to E-86; M-1 to L-85; M-1 to Y-84; M-1 to N-83;M-1 to W-82; M-1 to F-81; M-1 to Q-80; M-1 to T-79; M-1 to Y-78; M-1 toH-77; M-1 to R-76; M-1 to P-75; M-1 to P-74; M-1 to C-73; M-1 to P-72;M-1 to G-71; M-1 to C-70; M-1 to T-69; M-1 to T-68; M-1 to P-67; M-1 toS-66; M-1 to D-65; M-1 to R-64; M-1 to R-63; M-1 to C-62; M-1 to P-61;M-1 to R-60; M-1 to Q-59; M-1 to V-58; M-1 to F-57; M-1 to T-56; M-1 toG-55; M-1 to P-54; M-1 to P-53; M-1 to C-52; M-1 to Q-51; M-1 to A-50;M-1 to C-49; M-1 to V-48; M-1 to L47; M-1 to R-46; M-1 to E45; M-1 toG-44; M-1 to T43; M-1 to E42; M-1 to A-41; M-1 to D40; M-1 to R-39; M-1to W-38; M-1 to P-37; M-1 to Y-36; M-1 to T-35; M-1 to P-34; M-1 toT-33; M-1 to E-32; M-1 to A-31; M-1 to V-30; M-1 to G-29; M-1 to R-28;M-1 to V-27; M-1 to A-26; M-1 to P-25; M-1 to V-24; M-1 to P-23; M-1 toL-22; M-1 to L-21; M-1 to A-20; M-1 to P-19; M-1 to L-18; M-1 to A-17;M-1 to L-16; M-1 to V-15; M-1 to L-14; M-1 to C-13; M-1 to L-12; M-1 toL-11; M-1 to S-10; M-1 to L-9; M-1 to G-8; M-1 to P-7; and M-1 to G-6 ofthe sequence of the TNFR-6β shown in FIG. 2A (SEQ ID NO:4). Polypeptidesencoded by these polynucleotide fragments are also encompassed by theinvention.

The invention also provides polypeptides comprising or alternativelyconsisting of, one or more amino acids deleted from both the amino andthe carboxyl termini of a TNFR-6β polypeptide, which may be describedgenerally as having residues n²-m² of FIG. 2A (i.e., SEQ ID NO:4), wheren² and m² are integers as described above.

The present application is also directed to nucleic acid moleculescomprising, or alternatively, consisting of, a polynucleotide sequenceat least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to thepolynucleotide sequence encoding a TNFR polypeptide set forth herein asm-y, n-z, n¹-m¹, 30-m³, and/or n²-m². In preferred embodiments, theapplication is directed to nucleic acid molecules comprising, oralternatively, consisting of, a polynucleotide sequence at least 90%,92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequencesencoding polypeptides having the amino acid sequence of the specific N—and C-terminal deletions recited herein. The present invention alsoencompasses the above polynucleotide sequences fused to a heterologouspolynucleotide sequence. Polypeptides encoded by these nucleic acidsand/or polynucleotide sequences are also encompassed by the invention.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of a complete TNFR amino acid sequence encodedby a cDNA clone contained in ATCC Deposit No. 97810, or 97809, wherethis portion excludes from 1 to about 49 amino acids from the aminoterminus of the complete amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97810 and 97809, respectively, or from 1to about 107 or 58 amino acids from the carboxy terminus of the completeamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 97810 and 97809, respectively, or any combination of the above aminoterminal and carboxy terminal deletions, of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97810or 97809. Polypeptides encoded by all of the above polynucleotides arealso encompassed by the invention.

In addition to terminal deletion forms of the protein discussed above,it also will be recognized by one of ordinary skill in the art that someamino acid sequences of the TNFR polypeptides can be varied withoutsignificant effect on the structure or function of the proteins. If suchdifferences in sequence are contemplated, it should be remembered thatthere will be critical areas on the protein which determine activity.

Thus, the invention further includes variations of the TNFR polypeptideswhich show substantial TNFR polypeptide functional activity (e.g.,immunogenic activity, biological activity) or which include regions ofTNFR protein such as the protein portions discussed below. Such mutantsinclude deletions, insertions, inversions, repeats, and typesubstitutions selected according to general rules known in the art so ashave little effect on activity. For example, guidance concerning how tomake phenotypically silent amino acid substitutions is provided inBowie, J. U. et al., “Deciphering the Message in Protein Sequences:Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990),wherein the authors indicate that there are two main approaches forstudying the tolerance of an amino acid sequence to change. The firstmethod relies on the process of evolution, in which mutations are eitheraccepted or rejected by natural selection. The second approach usesgenetic engineering to introduce amino acid changes at specificpositions of a cloned gene and selections or screens to identifysequences that maintain functionality. As the authors state, thesestudies have revealed that proteins are surprisingly tolerant of aminoacid substitutions. The authors further indicate which amino acidchanges are likely to be permissive at a certain position of theprotein. For example, most buried amino acid residues require nonpolarside chains, whereas few features of surface side chains are generallyconserved. Other such phenotypically silent substitutions are describedin Bowie, J. U. et al., supra, and the references cited therein.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and Ile;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg and replacements amongthe aromatic residues Phe, Tyr. Thus, the fragment, derivative or analogof the polypeptide of SEQ ID NO:2, 4 or 6, or that encoded by adeposited cDNA, may be (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code, or(ii) one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature or solubleextracellular polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acids(such as, for example, an IgG Fc peptide fusion and/or an immunoglobulinlight chain constant region peptide), a leader or secretory sequence, ora sequence which is employed for purification of the TNFR polypeptide)are fused to a TNFR polypeptide described herein. Such fragments,derivatives and analogs are deemed to be within the scope of thoseskilled in the art from the teachings herein.

Thus, the TNFR of the present invention may include one or more aminoacid substitutions, deletions or additions, either from naturalmutations or human manipulation. As indicated, changes are preferably ofa minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein (seeTable III).

TABLE III Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Amino acids in the TNFR proteins of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for functionalactivity such as, for example, ligand/receptor (e.g., Fas ligand and/orAIM-II) receptor binding or in vitro or in vitro proliferative activity.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al, Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).

Replacement of amino acids can also change the selectivity of thebinding of a ligand to cell surface receptors. For example, Ostade etal., Nature 361:266-268 (1993) describes certain mutations resulting inselective binding of TNF-α to only one of the two known types of TNFreceptors. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

Since TNFR-6 alpha and TNFR-6 beta are members of the TNFreceptor-related protein family, to modulate rather than completelyeliminate biological activities of TNFR preferably mutations are made insequences encoding amino acids in the TNFR conserved extracellulardomain, more preferably in residues within this region which are notconserved among members of the TNF receptor family. Also forming part ofthe present invention are isolated polynucleotides comprising nucleicacid sequences which encode the above TNFR mutants.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of the TNFR polypeptides can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). Polypeptides of the invention also can bepurified from natural or recombinant sources using anti-TNFR-6 alpha andTNFR-6 beta antibodies of the invention in methods which are well knownin the art of protein purification.

The invention further provides isolated TNFR polypeptides comprising anamino acid sequence selected from the group consisting of: (a) the aminoacid sequence of a full-length TNFR polypeptide having the completeamino acid sequence shown in SEQ ID NO:2 or 4 or as encoded by the cDNAclone contained in the plasmid deposited as ATCC Deposit No. 97810 or97809; (b) the amino acid sequence of a mature TNFR polypeptide havingthe amino acid sequence at positions 31-300 in SEQ ID NO:2 or 31-170 inSEQ ID NO:4, or as encoded by the cDNA clone contained in the plasmiddeposited as ATCC Deposit No. 97810 or 97809; or (c) the amino acidsequence of a soluble extracellular domain of a TNFR polypeptide havingthe amino acid sequence at positions 31 to 283 in SEQ ID NO:2 or 31 to166 in SEQ ID NO:4, or as encoded by the cDNA clone contained in theplasmid deposited as ATCC Deposit No. 97810 or 97809.

Further polypeptides of the present invention include polypeptides whichhave at least 90% similarity, more preferably at least 80%, 85%, 90%,92%, or 95% similarity, and still more preferably at least 96%, 97%, 98%or 99% similarity to those described above. The polypeptides of theinvention also comprise those which are at least 80% identical, morepreferably at least 85%, 90%, 92% or 95% identical, still morepreferably at least 96%, 97%, 98% or 99% identical to the polypeptideencoded by the deposited cDNA (ATCC Deposit Nos. 97810 or 97809) or tothe polypeptide of SEQ ID NO:2 or 4, and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2:482489, 1981) tofnd the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a TNFR polypeptideis intended that the amino acid sequence of the polypeptide is identicalto the reference sequence except that the polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe reference amino acid of the TNFR polypeptide. In other words, toobtain a polypeptide having an amino acid sequence at least 80%, 85%,90%, or 95% identical to a reference amino acid sequence, up to 5% ofthe amino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the amino acid sequence shown in SEQ ID NO:2 or 4, or to anamino acid sequence encoded by the cDNA contained in the deposits havingATCC Deposit No. 97810, or 97809, or fragments thereof (e.g., thesequence of any of the polypeptides corresponding to N or C terminaldeletions of TNFR, as described herein (e.g., the polypeptide having thesequence of amino acids 30 to 300 of SEQ ID NO:2)) can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. According to this embodiment, if the subject sequence isshorter than the query sequence due to N— or C-terminal deletions, notbecause of internal deletions, a manual correction is made to theresults to take into consideration the fact that the FASTDB program doesnot account for N— and C-terminal truncations of the subject sequencewhen calculating global percent identity. For subject sequencestruncated at the N— and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N— and C-terminal of the subject sequence,which are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. A determination ofwhether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thisembodiment. Only residues to the N— and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N— andC-terminal residues of the subject sequence. For example, a 90 aminoacid residue subject sequence is aligned with a 100 residue querysequence to determine percent identity. The deletion occurs at theN-terminus of the subject sequence and therefore, the FASTDB alignmentdoes not show a matching/alignment of the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N— and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal deletionsso there are no residues at the N— or C-termini of the subject sequencewhich are not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N— and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected for. Noother manual corrections are made for the purposes of this embodiment.

The polypeptide of the present invention have uses which include, butare not limited to, as molecular weight markers on SDS-PAGE gels or onmolecular sieve gel filtration columns using methods well known to thoseof skill in the art. As described in detail below, the polypeptides ofthe present invention can also be used to raise polyclonal andmonoclonal antibodies, which are useful in assays for detecting TNFRprotein expression as described below or as agonists and antagonistscapable of enhancing or inhibiting TNFR protein function. Further, suchpolypeptides can be used in the yeast two-hybrid system to “capture”TNFR protein binding proteins which are also candidate agonists andantagonists according to the present invention. The yeast two hybridsystem is described in Fields and Song, Nature 340:245-246 (1989).

Transgenics

The proteins of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al, Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al, Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, Mol Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989));etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety. See also, U.S. Pat. No. 5,464,764 (Capecchi, etal., Positive-Negative Selection Methods and Vectors); U.S. Pat. No.5,631,153 (Capecchi, et al., Cells and Non-Human Organisms ContainingPredetermined Genomic Modifications and Positive-Negative SelectionMethods and Vectors for Making Same); U.S. Pat. No. 4,736,866 (Leder, etal., Transgenic Non-Human Animals); and U.S. Pat. No. 4,873,191 (Wagner,et al., Genetic Transformation of Zygotes); each of which is herebyincorporated by reference in its entirety. Further, the contents of eachof the documents recited in this paragraph is herein incorporated byreference in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)), each ofwhich is herein incorporated by reference in its entirety).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric animals. The transgene may be integrated as a single transgeneor as multiple copies such as in concatamers, e.g., head-to-head tandemsor head-to-tail tandems. The transgene may also be selectivelyintroduced into and activated in a particular cell type by following,for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl.Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences requiredfor such a cell-type specific activation will depend upon the particularcell type of interest, and will be apparent to those of skill in theart. When it is desired that the polynucleotide transgene be integratedinto the chromosomal site of the endogenous gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous geneare designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous gene. Thetransgene may also be selectively introduced into a particular celltype, thus inactivating the endogenous gene in only that cell type, byfollowing, for example, the teaching of Gu et al. (Science 265:103-106(1994)). The regulatory sequences required for such a cell-type specificinactivation will depend upon the particular cell type of interest, andwill be apparent to those of skill in the art. The contents of each ofthe documents recited in this paragraph is herein incorporated byreference in its entirety.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Female transgenic mice that secrete a TNFR-6 alpha and/or TNFR-6 betapolypeptide in their milk may be generated using the pBC1 MilkExpression Vector Kit, available from Invitrogen Corp. (Carlsbad,Calif.; Catalog Number K270-01). Transgenic mice can be made using thepBC I vector according to protocols well-known in the art. Milk may beharvested from the mice and TNFR-6 alpha and/or TNFR-6 beta polypeptidespurified from the milk according to the manufacturer's instructionspublished in the package insert that accompanies the pBC1 MilkExpression Vector Kit (Version B, 000829; 25-0264).

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of TNFR polypeptides, studyingconditions and/or disorders associated with aberrant TNFR expression,and in screening for compounds effective in ameliorating such conditionsand/or disorders.

In further embodiments of the invention, cells that are geneticallyengineered to express the proteins of the invention, or alternatively,that are genetically engineered not to express the proteins of theinvention (e.g., knockouts) are administered to a patient in vivo. Suchcells may be obtained from the patient (i.e., animal, including human)or an MHC compatible donor and can include, but are not limited tofibroblasts, bone marrow cells, blood cells (e.g., lymphocytes),adipocytes, muscle cells, endothelial cells, etc. The cells aregenetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally. Alternatively, the cells can be incorporated into amatrix and implanted in the body, e.g., genetically engineeredfibroblasts can be implanted as part of a skin graft; geneticallyengineered endothelial cells can be implanted as part of a lymphatic orvascular graft. (See, for example, Anderson et al. U.S. Pat. No.5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959, each of whichis incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Antibodies

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which immunospecifically bind a polypeptide, preferablyan epitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.In a preferred embodiment, the immunoglobulin is an IgG1 isotype. Inanother preferred embodiment, the immunoglobulin is an IgG4 isotype.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, shiprabbit, goat, guinea pig, camel, horse, or chicken. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulin and that do not express endogenous immunoglobulins,as described infra and, for example in, U.S. Pat. No. 5,939,598 byKucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention that they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies thatspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. Antibodies that do not bind polypeptides with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%,identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. Further included in the present invention areantibodies that bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, or 10⁻⁵M. More preferred binding affinities include those with a dissociationconstant or Kd less than 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M, or10⁻⁸ M. Even more preferred binding affinities include those with adissociation constant or Kd less than 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, ¹⁰⁻¹² M, 5×10⁻¹³ M, 10¹³ M, 5×10⁻¹⁴ M,10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand or receptor activity by at least 90%,at least 80%, at least 70%, at least 60%, or at least 50% of theactivity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation. The antibodiesmay be specified as agonists, antagonists or inverse agonists forbiological activities comprising the specific biological activities ofthe peptides of the invention disclosed herein. The above antibodyagonists can be made using methods known in the art. See, e.g., PCTpublication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood92(6):1981-1988 (1998); Chen, et al., Cancer Res. 58(16):3668-3678(1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al.,Cancer Res. 58(15):3209-3214 (1998); Yoon, et al., J. Immunol.160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247(1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997);Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol.Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762(1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al.,Cytokine 8(1):14-20 (1996) (which are all incorporated by referenceherein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

By way of another non-limiting example, antibodies of the invention maybe administered to individuals as a form of passive immunization.Alternatively, antibodies of the present invention may be used forepitope mapping to identify the epitope(s) bound by the antibody.Epitopes identified in this way may, in turn, for example, be used asvaccine candidates, i.e., to immunize an individual to elicit antibodiesagainst the naturally occuring forms of TNFR-6 alpha and/or TNFR-6 beta.

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N— or C-terminus or chemicallyconjugated (including covalent and non-covalent conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, or toxins. See, e.g., PCT publicationsWO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP396,387. Additional antibodies of the invention may be to albumin, asdescribed above.

The antibodies of the invention include derivatives that are modified,i.e, by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well-known in the art and arediscussed in detail in Example 11. Briefly, mice can be immunized with apolypeptide of the invention or a cell expressing such peptide. Once animmune response is detected, e.g., antibodies specific for the antigenare detected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well-knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the ATCC. Hybridomas are selected and cloned bylimited dilution. The hybridoma clones are then assayed by methods knownin the art for cells that secrete antibodies capable of binding apolypeptide of the invention. Ascites fluid, which generally containshigh levels of antibodies, can be generated by immunizing mice withpositive hybridoma clones.

Another well known method for producing both polyclonal and monoclonalhuman B cell lines is transformation using Epstein Barr Virus (EBV).Protocols for generating EBV-transformed B cell lines are commonly knownin the art, such as, for example, the protocol outlined in Chapter 7.22of Current Protocols in Immunology, Coligan et al., Eds., 1994, JohnWiley & Sons, NY, which is hereby incorporated in its entirety byreference herein. The source of B cells for transformation is commonlyhuman peripheral blood, but B cells for transformation may also bederived from other sources including, but not limited to, lymph nodes,tonsil, spleen, tumor tissue, and infected tissues. Tissues aregenerally made into single cell suspensions prior to EBV transformation.Additionally, steps may be taken to either physically remove orinactivate T cells (e.g., by treatment with cyclosporin A) in Bcell-containing samples, because T cells from individuals seropositivefor anti-EBV antibodies can suppress B cell immortalization by EBV. Ingeneral, the sample containing human B cells is innoculated with EBV,and cultured for 3-4 weeks. A typical source of EBV is the culturesupernatant of the B95-8 cell line (ATCC #VR-1492). Physical signs ofEBV transformation can generally be seen towards the end of the 3-4 weekculture period. By phase-contrast microscopy, transformed cells mayappear large, clear, hairy and tend to aggregate in tight clusters ofcells. Initially, EBV lines are generally polyclonal. However, overprolonged periods of cell cultures, EBV lines may become monoclonal orpolyclonal as a result of the selective outgrowth of particular B cellclones. Alternatively, polyclonal EBV transformed lines may be subcloned(e.g., by limiting dilution culture) or fused with a suitable fusionpartner and plated at limiting dilution to obtain monoclonal B celllines. Suitable fusion partners for EBV transformed cell lines includemouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma celllines (human x mouse; e.g., SPAM-8, SBC-H20, and CB-F7), and human celllines (e.g., GM 1500, SKO-007, RPM1 8226, and KR4). Thus, the presentinvention also provides a method of generating polyclonal or monoclonalhuman antibodies against polypeptides of the invention or fragmentsthereof, comprising EBV-transformation of human B cells.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entireties. Humanized antibodies areantibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska at al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat.Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; 5,939,598; 6,075,181; and 6,114,598, which are incorporatedby reference herein in their entirety. In addition, companies such asAbgenix, Inc. (Freemont, Calif.) and Genpharrn (San Jose, Calif.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby activate orblock TNFR mediated inhibition of apoptosis.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:2 or 4.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be obtained from a suitable source (e.g., an antibodycDNA library, or a cDNA library generated from, or nucleic acid,preferably poly A+RNA, isolated from, any tissue or cells expressing theantibody, such as hybridoma cells selected to express an antibody of theinvention) by PCR amplification using synthetic primers hybridizable tothe 3′ and 5′ ends of the sequence or by cloning using anoligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties ), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Nat. Acad. Sci. 81:851-855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-54) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,1988, Science 242:1038-1041).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques. Methodsof producing antibodies include, but are not limited to, hybridomatechnology, EBV transformation, and other methods discussed herein aswell as through the use recombinant DNA technology, as discussed below.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, e.g., a heavy or light chain of anantibody of the invention, requires construction of an expression vectorcontaining a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule or a heavy or light chainof an antibody, or portion thereof (preferably containing the heavy orlight chain variable domain), of the invention has been obtained, thevector for the production of the antibody molecule may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the invention, or a heavy orlight chain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; Cockett et al., 1990,Bio/Technology 8:2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa califomica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiationsignals may also be required for efficient translation of insertedantibody coding sequences. These signals include the ATG initiationcodon and adjacent sequences. Furthermore, the initiation codon must bein phase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bittner et al., 1987, Methods inEnzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215); and hygro,which confers resistance to hygromycin (Santerre et al., 1984, Gene30:147). Methods commonly known in the art of recombinant DNA technologywhich can be used are described in Ausubel et al. (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CurrentProtocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin etal., 1981, J. Mol. Biol. 150:1, which are incorporated by referenceherein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. An advantage of glutaminesynthase based vectors are the availabilty of cell lines (e.g., themurine myeloma cell line, NS0) which are glutamine synthase negative. Itis also possible to amplify vectors that utilize glutamine synthaseselection in glutamine synthase expressing cells (e.g., Chinese HamsterOvary (CHO) cells), however, by providing additional inhibitor toprevent the functioning of the endogenous gene. A glutamine synthaseexpression system and components thereof are detailed in PCTpublications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; andWO91/06657 which are hereby incorporated in their entireties byreference herein. Additionally, glutamine synthase expression vectorscan be obtained from Lonza Biologics, Inc. (Portsmouth, N.H.).Expression and production of monoclonal antibodies using a GS expressionsystem in murine myeloma cells is described in Bebbington et al.,Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog.11:1 (1995) which are herein incorporated by reference.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci.USA 77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

Antibody conjugates

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20 or 50 amino acids of the polypeptide) of the present invention togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. The antibodies may bespecific for antigens other than polypeptides (or portion thereof,preferably at least 10, 20 or 50 amino acids of the polypeptide) of thepresent invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

As discussed, supra, the polypeptides of the present invention may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides of the presentinvention may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide- linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995)0.

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitates theirpurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the FLAG® tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. See,for example, U.S. Pat. No. 4,741,900 for metal ions which can beconjugated to antibodies for use as diagnostics according to the presentinvention. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (35S),tritium (³H), indium (¹¹¹In, ¹¹²In, ^(113m)In, ^(115m)In), technetium(⁹⁹Tc,^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³ Xe), fluorine (¹⁸F), ¹⁵³Sm,¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re,¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi or other radioisotopes suchas, for example, ¹⁰³Pd, ¹³³Xe, 131I, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S,⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, ⁹⁰Y, ¹¹⁷Tin, ¹⁸⁶Re,¹⁸⁸Re and ¹⁶⁶Ho. In specific embodiments, an antibody or fragmentthereof is attached to macrocyclic chelators useful for chelatingradiometal ions, including but not limited to, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and¹⁵³Sm, to polypeptides. In a preferred embodiment, the radiometal ionassociated with the macrocyclic chelators attached to antibodies of theinvention is ¹¹¹In. In another preferred embodiment, the radiometal ionassociated with the macrocyclic chelator attached to antibodies of theinvention is ⁹⁰Y. In specific embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inother specific embodiments, the DOTA is attached to the an antibody ofthe invention or fragment thereof via a linker molecule. Examples oflinker molecules useful for conjugating DOTA to a polypeptide arecommonly known in the art—see, for example, DeNardo et al., Clin CancerRes. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7,1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999 which arehereby incorporated by reference in their entirety. In addition U.S.Pat. Nos. 5,652,361 and 5,756,065, which disclose chelating agents thatmay be conjugated to antibodies, and methods for making and using them,are hereby incorporated by reference in their entireties.

A cytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis- dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Techniques known in the art may be applied to label polypeptides andantibodies (as well as fragments and variants of polypetides andantibodies) of the invention. Such techniques include, but are notlimited to, the use of bifunctional conjugating agents (see e.g., U.S.Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931;5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and5,808,003; the contents of each of which are hereby incorporated byreference in its entirety) and direct coupling reactions (e.g.,Bolton-Hunter and Chloramine-T reaction).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest is conjugated to a labeled compound (e.g., 3H or125I) in the presence of increasing amounts of an unlabeled secondantibody.

Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the described disorders. Therapeutic compounds of theinvention include, but are not limited to, antibodies of the invention(including fragments, analogs and derivatives thereof as describedherein) and nucleic acids encoding antibodies of the invention(including fragments, analogs and derivatives thereof as describedherein). The antibodies of the invention can be used to treat or preventdiseases and disorders associated with aberrant expression and/oractivity of a polypeptide of the invention, including, but not limitedto, diseases and/or disorders such as autoimmune diseases and/ordeficiencies, as discussed herein. The treatment and/or prevention ofdiseases and disorders associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases and disorders.Antibodies of the invention may be provided in pharmaceuticallyacceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy, anti-retroviral agents,and anti-tumor agents). Generally, administration of products of aspecies origin or species reactivity (in the case of antibodies) that isthe same species as that of the patient is preferred. Thus, in apreferred embodiment, human antibodies, fragments derivatives, analogs,or nucleic acids, are administered to a human patient for therapy orprophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides,including fragments thereof. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10-6 M, 10-6 M, 5×10-7 M,10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5×10-11M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M,5×10-15 M, and 10-15 M.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody nucleic acids(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935;Zijlstra et al., 1989, Nature 342:435-438). In specific embodiments, theexpressed antibody molecule is a single chain antibody; alternatively,the nucleic acid sequences include sequences encoding both the heavy andlight chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid- carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180 dated April 16, 1992(Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO 92/20316 dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993(Clarke et al.), WO 93/20221 dated Oct. 14, 1993 (Young)).Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., 1993, Meth. Enzymol.217:581-599). These retroviral vectors have been to delete retroviralsequences that are not necessary for packaging of the viral genome andintegration into host cell DNA. The nucleic acid sequences encoding theantibody to be used in gene therapy are cloned into one or more vectors,which facilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., 1994, Biotherapy6:291-302, which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin.Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons andGunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson,1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431434; Rosenfeld et al., 1992, Cell 68:143-155; Mastrangeliet al., 1993, J. Clin. Invest. 91:225-234; PCT Publication WO94/12649;and Wang, et al., 1995, Gene Therapy 2:775-783. In a preferredembodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300;U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598, dated Apr. 28,1994; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980,Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction include butare not limited to intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes. Thecompounds or compositions may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceutical compoundsor compositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, 1990, Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201;Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J.Med. 321:574). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y.(1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem.23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989,Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosising a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Assaying TR6-alpha and/or TR6-beta polypeptide levels in a biologicalsample can occur using antibody-based techniques. Antibodies of theinvention can be used to assay protein levels in a biological sampleusing classical immunohistological methods known to those of skill inthe art (e.g., see Jalkanen, M., et al., J. Cell. Biol. 101:976-985(1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096 (1987)).Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I),carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In,^(112m)In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti),gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon(¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb,¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminescent labels,such as luminol; and fluorescent labels, such as fluorescein andrhodamine, and biotin.

Techniques known in the art may be applied to label antibodies of theinvention. Such techniques include, but are not limited to, the use ofbifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065;5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contentsof each of which are hereby incorporated by reference in its entirety).

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide of theinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface- bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Immune System-Related Disorders

Diagnosis

The present inventors have discovered that TNFR-6 alpha and TNFR-6 betaare expressed in hematopoietic and transformed tissues. For a number ofimmune system-related disorders, substantially altered (increased ordecreased) levels of TNFR gene expression can be detected in immunesystem tissue or other cells or bodily fluids (e.g., sera and plasma)taken from an individual having such a disorder, relative to a“standard” TNFR gene expression level, that is, the TNFR expressionlevel in immune system tissues or other cells or bodily fluids from anindividual not having the immune system disorder. Thus, the inventionprovides a diagnostic method useful during diagnosis of an immune systemdisorder, which involves measuring the expression level of the geneencoding the TNFR protein in immune system tissue or other cells or bodyfluid from an individual and comparing the measured gene expressionlevel with a standard TNFR gene expression level, whereby an increase ordecrease in the gene expression level compared to the standard isindicative of an immune system disorder.

In particular, it is believed that certain tissues in mammals withcancer (e.g., colon, breast and lung cancers) have elevated copy numbersof TNFR genes and/or express significantly elevated levels of the TNFRprotein and mRNA encoding the TNFR when compared to a corresponding“standard” level. Further, it is believed that elevated levels of theTNFR protein can be detected in certain cells or body fluids (e.g., seraand plasma) from mammals with such a cancer when compared to sera frommammals of the same species not having the cancer.

Thus, the invention provides a diagnostic method useful during diagnosisof an immune system disorder, including cancers which involves measuringthe expression level of the gene encoding the TNFR protein in immunesystem tissue or other cells or body fluid from an individual andcomparing the measured gene expression level with a standard TNFR geneexpression level, whereby an increase or decrease in the gene expressionlevel compared to the standard is indicative of an immune systemdisorder.

Where a diagnosis of a disorder in the immune system including diagnosisof a tumor has already been made according to conventional methods, thepresent invention is useful as a prognostic indicator, whereby patientsexhibiting depressed gene expression will experience a worse clinicaloutcome relative to patients expressing the gene at a level nearer thestandard level.

By “assaying the expression level of the gene encoding a TNFR protein”is intended qualitatively or quantitatively measuring or estimating thelevel of the TNFR-6α and/or TNFR-6β protein or the level of the mRNAencoding the TNFR-6α and/or TNFR-6β protein in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to the TNFRprotein level or mRNA level in a second biological sample). Preferably,the TNFR protein level or mRNA level in the first biological sample ismeasured or estimated and compared to a standard TNFR protein level ormRNA level, the standard being taken from a second biological sampleobtained from an individual not having the disorder or being determinedby averaging levels from a population of individuals not having adisorder of the immune system. As will be appreciated in the art, oncestandard TNFR protein levels or mRNA levels are known, they can be usedrepeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains TNFR protein or mRNA. As indicated, biological samplesinclude body fluids (such as sera, plasma, urine, synovial fluid andspinal fluid) which contain free extracellular domain(s) (or solubleform(s)) of a TNFR protein, immune system tissue, and other tissuesources found to express complete TNFR, mature TNFR, or extracellulardomain of a TNFR. Methods for obtaining tissue biopsies and body fluidsfrom mammals are well known in the art. Where the biological sample isto include mRNA, a tissue biopsy is the preferred source.

The invention also contemplates the use of a gene of the presentinvention for diagnosing mutations in a TNFR gene. For example, if amutation is present in one of the genes of the present invention,conditions would result from a lack of production of the receptorpolypeptides of the present invention. Further, mutations which enhancereceptor polypeptide activity would lead to diseases associated with anover expression of the receptor polypeptide, e.g., cancer. Mutations inthe genes can be detected by comparing the sequence of the defectivegene with that of a normal one. Subsequently one can verify that amutant gene is associated with a disease condition or the susceptibilityto a disease condition. That is, a mutant gene which leads to theunderexpression of the receptor polypeptides of the present inventionwould be associated with an inability of TNFR to inhibit Fas ligandand/or AIM-II mediated apoptosis, and thereby result in irregular cellproliferation (e.g., tumor growth).

Other immune system disorders which may be diagnosed by the foregoingassays include, but are not limited to, hypersensitivity, allergy,infectious disease, graft-host disease, Immunodificiency, autoimmunediseases and the like.

Individuals carrying mutations in the genes of the present invention maybe detected at the DNA level by a variety of techniques. Nucleic acidsused for diagnosis may be obtained from a patient's cells, such as fromblood, urine, saliva and tissue biopsy among other tissues. The genomicDNA may be used directly for detection or may be amplified enzymaticallyby using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior toanalysis. RNA or cDNA may also be used for the same purpose. As anexample, PCR primers complementary to the nucleic acid of the instantinvention can be used to identify and analyze mutations in the humangenes of the present invention. For example, deletions and insertionscan be detected by a change in the size of the amplified product incomparison to the normal genotype. Point mutations can be identified byhybridizing amplified DNA to radiolabeled RNA or alternatively,radiolabeled antisense DNA sequences of the present invention. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNaseA digestion or by differences in melting temperatures. Such a diagnosticwould be particularly useful for prenatal or even neonatal testing.

Sequence differences between the reference gene and “mutants” may berevealed by the direct DNA sequencing method. In addition, cloned DNAsegments may be used as probes to detect specific DNA segments. Thesensitivity of this method is greatly enhanced when combined with PCR.For example, a sequencing primer used with double stranded PCR productor a single stranded template molecule generated by a modified PCRproduct. The sequence determination is performed by conventionalprocedures with radiolabeled nucleotides or by automatic sequencingprocedures with fluorescent tags.

Sequence changes at the specific locations may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (for example, Cotton et al., PNAS, 85:4397-4401 (1985)).

Assaying TNFR protein levels in a biological sample can occur usingantibody-based techniques. For example, TNFR protein expression intissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-basedmethods useful for detecting TNFR gene expression include immunoassays,such as the enzyme linked immunosorbent assay (ELISA) and theradioimmunoassay (RIA). Suitable antibody assay labels are known in theart and include enzyme labels, such as, glucose oxidase, andradioisotopes, such as iodine (125I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹²In), and technetium (^(99m)Tc), and fluorescentlabels, such as fluorescein and rhodamine, and biotin.

In addition to assaying TNFR protein levels in a biological sampleobtained from an individual, TNFR proteins can also be detected in vivoby imaging. Antibody labels or markers for in vivo imaging of TNFRproteins include those detectable by X-radiography, NMR or ESR. ForX-radiography, suitable labels include radioisotopes such as barium orcesium, which emit detectable radiation but are not overtly harmful tothe subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

A TNFR-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, ¹³¹I, ¹¹²In, ^(99m)Tc, (¹³¹I, 125I, 123I, ¹²¹I), carbon(¹⁴C), sulfur (³⁵S), tritium (3H), indium (^(115m)In, ^(113m)In, ¹¹²In,111In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium(⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe),fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y,⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, 105Rh, ⁹⁷Ru), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously or intraperitoneally) into themammal to be examined for inunune system disorder. It will be understoodin the art that the size of the subject and the imaging system used willdetermine the quantity of imaging moiety needed to produce diagnosticimages. In the case of a radioisotope moiety, for a human subject, thequantity of radioactivity injected will normally range from about 5 to20 millicuries of ^(99m)Tc. The labeled antibody or antibody fragmentwill then preferentially accumulate at the location of cells whichcontain TNFR protein. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982)).

Treatment

The Tumor Necrosis Factor (TNF) family ligands are known to be among themost pleiotropic cytokines, inducing a large number of cellularresponses, including cytotoxicity, anti-viral activity, immunoregulatoryactivities, and the transcriptional regulation of several genes(Goeddel, D. V. et al., “Tumor Necrosis Factors: Gene Structure andBiological Activities,” Symp. Quant. Biol. 51:597-609 (1986), ColdSpring Harbor; Beutler, B., and Cerami, A., Annu. Rev. Biochem.57:505-518 (1988); Old, L. J., Sci. Am. 258:59-75 (1988); Fiers, W.,FEBS Lett. 285:199-224 (1991)). The TNF-family ligands induce suchvarious cellular responses by binding to TNF-family receptors.

TNFR-6 alpha and/or TNFR-6 beta polynucleotides and polypeptides of theinvention may be used in developing treatments for any disorder mediated(directly or indirectly) by defective, or insufficient amounts of TNFR-6alpha and/or TNFR-6 beta. TNFR-6 alpha and/or TNFR-6 beta polypeptidesmay be administered to a patient (e.g., mammal, preferably human)afflicted with such a disorder. Alternatively, a gene therapy approachmay be applied to treat such disorders. Disclosure herein of TNFR-6alpha and/or TNFR-6 beta nucleotide sequences permits the detection ofdefective TNFR-6 alpha and/or TNFR-6 beta genes, and the replacementthereof with normal TNFR-6 alpha and/or TNFR-6 beta -encoding genes.Defective genes may be detected in in vitro diagnostic assays, and bycomparison of a TNFR-6 alpha and/or TNFR-6 beta nucleotide sequencedisclosed herein with that of a TNFR-6 alpha and/or TNFR-6 beta genederived from a patient suspected of harboring a defect in this gene.

In another embodiment, the polypeptides of the present invention areused as a research tool for studying the biological effects that resultfrom inhibiting Fas ligand/TNFR-6 alpha and/or TNFR-6 beta and/or AIM-IIinteractions on different cell types. TNFR-6 alpha and/or TNFR-6 betapolypeptides also may be employed in in vitro assays for detecting Fasligand, AIM-II, or TNFR-6 alpha and/or TNFR-6 beta or the interactionsthereof.

In another embodiment, a purified TNFR-6 alpha and/or TNFR-6 betapolypeptide of the invention is used to inhibit binding of Fas ligandand/or AIM-II to endogenous cell surface Fas ligand and/or AIM-IIreceptors. Certain ligands of the TNF family (of which Fas ligand andAIM-II are members) have been reported to bind to more than one distinctcell surface receptor protein. AIM-II likewise is believed to bindmultiple cell surface proteins. By binding Fas ligand and/or AIM-II,soluble TNFR-6 alpha and/or TNFR-6 beta polypeptides of the presentinvention may be employed to inhibit the binding of Fas ligand and/orAIM-II not only to endogenous TNFR-6 alpha and/or TNFR-6 beta, but alsoto Fas ligand and AIM-II receptor proteins that are distinct from TNFR-6alpha and/or TNFR-6 beta. Thus, in another embodiment, TNFR-6 alphaand/or TNFR-6 beta is used to inhibit a biological activity of Fasligand and/or AIM-II, in in vitro or in vivo procedures. By inhibitingbinding of Fas ligand and/or AIM-II to cell surface receptors, TNFR-6alpha and/or TNFR-6 beta polypeptides of the invention also inhibitbiological effects that result from the binding of Fas ligand and/orAIM-II to endogenous receptors. Various forms of TNFR-6 alpha and/orTNFR-6 beta may be employed, including, for example, the above-describedTNFR-6 alpha and/or TNFR-6 beta fragments, derivatives, and variantsthat are capable of binding Fas ligand and/or AIM-II. In a preferredembodiment, a soluble TNFR-6 alpha and/or TNFR-6 beta polypeptide of theinvention is administered to inhibit a biological activity of Fas ligandand/or AIM-II, e.g., to inhibit Fas ligand-mediated and/orAIM-II-mediated apoptosis of cells susceptible to such apoptosis.

In a further embodiment, a TNFR-6 alpha and/or TNFR-6 beta polypeptideof the invention is administered to a mammal to treat a Fasligand-mediated and/or AIM-II-mediated disorder. Such Fasligand-mediated and/or AIM-II-mediated (e.g., a human) disorders includeconditions caused (directly or indirectly) or exacerbated by Fas ligandand/or AIM-II.

There are numerous autoimmune diseases in which FasL/Fas interactionsplay a role. In patients experiencing GVHD, serum levels of FasL wereabnormally high as was the number of FasL⁺ T cells . The CNS plaquesfrom patients with MS have been shown to express high levels of Fas andFasL. This is particularly significant since Fas and FasL expression isnormally absent in the mature CNS. As with NOD mice, patients with IDDMhave a superabundance of FasL⁺ T cells associated with their isletcells. As evidence of FasL/Fas mediated cell killing, patients withchronic renal failure have been reported to have a 50 fold increase inthe number of apoptotic nephrons compared to normal. This has beenascribed to renal tubule epithelial cell expression of both FasL andFas, leading to cellular fratricide . In the joints of rheumatoidarthritic patients, activated T cells expressing FasL are seen inconjunction with Fas expressing chondrocytes. In ulcerative colitis(UC), Fas expression is observed on colonic epithelial cells, and FasLon lamina propria lymphocytes. This lead to the observation that FasLpositive lymphocytes are present only in the lamina propria of UCpatients with active lesions but not in tissues from inactive UCpatients.

Two clinical indications in which the role of FasL-mediated killing ismost apparent are myelodisplastic syndrome (MDS) and the neutropeniaassociated with large granular lymphocyte (LGL) leukemia. In MDS, bonemarrow hematopoetic cells suffer an abnormally high level of apoptosis,associated with the upregulation of bone marrow Fas expression andlymphocyte FasL expression. The neutropenia seen in patients with LGLleukemia has been attributed to the high levels of circulating serumFasL. When leukemic LGL serum was incubated in vitro for 24 hours withnormal neutrophils, the degree of apoptosis significantly increasedabove that of cells incubated with normal serum.

As described in detail in Example 22, below, TNFR6-Fc is a potentinhibitor FasL-mediated killing. Thus, the FasL-associated disorderslisted above may be treated and/or prevented, in accordance with theinvention, through administration of the TNFR6-containing polypeptidesand polynucleotides desribed herein.

Suitable animal models for examining the effectiveness of TNFR6 intreating disease include but are not limited to mouse models of graftversus host disease (GVHD), murine allergic encephalomyelitis (EAE), anassay used as a central nervous system (CNS) model of multiple sclerosis(MS); non-obese diabetic (NOD) mouse model of insulin-dependant diabetesmellitus (IDDM), which is characterized by FasL⁺ T cell destruction ofislet cells, while Fas⁻ NOD mice fail to develop diabetes. NOD mice canalso be used to model Sjogren's disease, since apoptosis in the salivaryand lacrimal glands of these mice has been reported. In a mouse model ofchronic renal failure, ROP-Os/+ mice developed spontaneous tubularatrophy and renal failure correlated with upregulation of Fas and FasLin these tissues. The invention encompasses the treatment and preventionof the human disesases corresonding to these animal models, throughadministration of the TNFR6 polypeptides and polynucleotides of thepresent invention.

In addition, TNFR6 binds to LIGHT (TL3), a regulator of T cell funtion.As detailed in Example 23, below, TNFR6-Fc can ameliorate the effects oftransplantation, including the inhibition of transplant or graftrejection and the inhibition of graft versus host disease (GVHD). Themethods encompass the treatment of graft rejection or GVHD wherein thegrafted tissue or organ is one or more of a variety of tissues and/ororgans, including, but not limited to, heart, lung, kidney, liver,pancreas, islet cells, bone marrow, and skin. Such methods of preventingFasL-mediated killing or ameliorating the effects of transplantation maybe carried out, in accordance with the present invention, usingTNFR6-human serum albumin fusions, in lieu of Fc fusions.

Cells which express a TNFR polypeptide and have a potent cellularresponse to TNFR-6α and TNFR-6βligands include lymphocytes, endothelialcells, keratinocytes, and prostate tissue. By “a cellular response to aTNF-family ligand” is intended any genotypic, phenotypic, and/ormorphologic change to a cell, cell line, tissue, tissue culture orpatient that is induced by a TNF-family ligand. As indicated, suchcellular responses include not only normal physiological responses toTNF-family ligands, but also diseases associated with increasedapoptosis or the inhibition of apoptosis. Additionally, as describedherein, TNFR polypeptides of the invention bind Fas ligand and AIM-IIand consequently block Fas ligand and AIM-II mediated apoptosis.Apoptosis-programmed cell death is a physiological mechanism involved inthe deletion of B and/or T lymphocytes of the immune system, and itsdisregulation can lead to a number of different pathogenic processes (J.C. Ameisen AIDS 8:1197-1213 (1994); P. H. Kramner et al., Curr. Opin.Immunol. 6:279-289 (1994)).

Diseases associated with increased cell survival, or the inhibition ofapoptosis, include cancers (such as follicular lymphomas, carcinomaswith p53 mutations, and hormone-dependent tumors, including, but notlimited to colon cancer, cardiac tumors, pancreatic cancer, melanoma,retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicularcancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Grave's disease, Hashimoto's thyroiditis,autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn'sdisease, polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis (e.g., proliferative glomerulonephritis), autoimmunegastritis, autoimmune thrombocytopenic purpura, and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft vs. host disease (acute and/orchronic), acute graft rejection, and chronic graft rejection. Inpreferred embodiments, TNFR polynucleotides, polypeptides, and/orantagonists of the invention are used to inhibit growth, progression,and/or metastasis of cancers, in particular those listed above.

Additional diseases or conditions associated with increased cellsurvival include, but are not limited to, progression, and/or metastasesof malignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with increased apoptosis include AIDS;neurodegenerative disorders (such as Alzheimer's disease, Parkinson'sdisease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellardegeneration and brain tumor or prior associated disease); autoimmunedisorders (such as, multiple sclerosis, Sjogren's syndrome, Grave'sdisease Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis,Behcet's disease, Crohn's disease, polymyositis, systemic lupuserythematosus, immune-related glomerulonephritis (e.g., proliferativeglomerulonephritis), autoimmune gastritis, thrombocytopenic purpura, andrheumatoid arthritis) myelodysplastic syndromes (such as aplasticanemia), graft vs. host disease (acute and/or chronic), ischemic injury(such as ischemic cardiac injury and that caused by myocardialinfarction, stroke and reperfusion injury), liver injury or disease(e.g., hepatitis related liver injury, cirrhosis, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, ulcerativecolitis, cachexia and anorexia. In preferred embodiments, TNFRpolynucleotides, polypeptides and/or agonists are used to treat orprevent the diseases and disorders listed above.

In a specific embodiment, TNFR polynucleotides, polypeptides, oragonists of the invention are used to treat and/or preventglomerulonephritis. In a further embodiment, TNFR polynucleotides,polypeptides, or agonists of the invention are used to treat and/orprevent chronic glomerulonephritis and/or cell/tissue damage (e.g.,glomerular cell death) and/or medical conditions associated with thisdisease. In a further nonexclusive embodiment, TNFR polynucleotides,polypeptides, or agonists of the invention are used to treat and/orprevent proliferative glomerulonephritis and/or cell/tissue damage(e.g., glomerular cell death) and/or medical conditions associated withthis disease.

In a specific embodiment, TNFR polynucleotides, polypeptides, oragonists of the invention are used treat or prevent biliary cirrhosisand/or medical conditions associated with this disease.

In a specific embodiment, TNFR polynucleotides, polypeptides, oragonists of the invention are used treat or prevent disease, such as,for example, alcoholic liver disease and/or medical conditionsassociated with this disease (e.g., cirrhosis).

In a specific embodiment, TNFR polynucleotides, polypeptides, oragonists of the invention are used to treat and/or prevent graft vs hostdisease. In a specific embodiment TNFR polynucleotides, polypeptides, oragonists of the invention are used to treat (e.g., reduce) or preventtissue or cell damage or destruction (e.g., lymphoid cell depletionassociated with graft vs host disease) and/or other medical conditionsassociated with this disease. In another non exclusive specificembodiment, the TNFR polynucleotides, polypeptides, or agonists of theinvention are used to treat (e.g., reduce) and/or prevent diarrheaduring graft vs host disease.

In a specific embodiment, TNFR polynucleotides, polypeptides, and/oragonists or antagonists of the invention are used to treat and/orprevent Sjogren's diesease and/or to reduce tissue/cell damage ordestruction (e.g., damage or destruction of salivary and/or lacrimaltissues) and/or other medical conditions associated with this disease.

In a specific embodiment, TNFR polynucleotides, polypeptides, oragonists of the invention are used to treat and/or prevent multiplesclerosis and/or to reduce tissue damage or destruction (such as, forexample, neurological tissue (e.g., CNS tissue) damage or destruction)and/or lesions or other medical conditions associated with this disease.

In a specific embodiment, TNFR polynucleotides, polypeptides, oragonists, including antibody and antibody fragments, of the inventionare used to treat and/or prevent Alzheimer's disease and/or to reducetissue damage or destruction (e.g., damage or destruction ofneurological tissue or cells) and/or medical conditions associated withthis disease.

In a specific embodiment, TNFR polynucleotides, polypeptides, oragonists of the invention are used to treat, prevent Parkinson's diseaseand/or to reduce tissue damage or destruction (e.g., damage ordestruction of neurological tissue or cells, such as, for exampleneuronal cells) and/or medical conditions associated with this disease.

In a specific embodiment, TNFR polynucleotides, polypeptides, oragonists of the invention are used before, during, immediately after,and/or after a stroke to treat, prevent, or reduce damage of cells ortissue (such as, for example, neurological tissue) and/or medicalconditions associated with stroke.

In a specific embodiment, TNFR polynucleotides, polypeptides, oragonists of the invention are used to treat, prevent, or reduce ischemicinjury (such as, for example, ischemic cardiac injury) and/or medicalconditions associated with ischemic injury. In a specific embodiment,TNFR polynucleotides, polypeptides, or agonists of the invention areused before, during, immediately after, and/or after a heart attack totreat, prevent, or reduce ischemic cardiac injury.

In another specific embodiment, TNFR polynucleotides, polypeptides,and/or agonists of the invention are used to treat or preventmyelodysplastic syndromes (MDS) and/or medical conditions associatedwith MDS.

In another specific embodiment, TNFR polynucleotides, polypeptides, oragonists of the invention are used to increase circulating blood cellnumbers in patients suffering from cytopenia, lymphopenia and/or anemia.

In a specific embodiment, TNFR polynucleotides, polypeptides, oragonists of the invention are used to treat and/or prevent Hashimoto'sthyroiditis and/or to reduce destruction or damage of tissue or cells(e.g., thyroid gland) and/or to treat or prevent medical conditionsassociated with this disease.

In a specific embodiment, TNFR polynucleotides, polypeptides, oragonists of the invention are used to treat (e.g., reduce) and/orprevent autoimmune gastritis and/or medical conditions associated withthis disease.

In a specific embodiment, TNFR polynucleotides, polypeptides, oragonists of the invention are used to treat and/or prevent ulcerativecolitis and/or cell/tissue damage (e.g., ulceration in the colon) and/ormedical conditions associated with this disease.

In a specific embodiment, TNFR polynucleotides, polypeptides, and/oragonists or antagonists of the invention are used to treat and/orprevent rheumatoid arthritis and/or medical conditions associated withthis disease.

Additionally, a number of cancers secrete FasL which binds Fas positiveT cells and kills them. Any cancer which expresses FasL could thereforbe a target for treatment by TNFR and TNFR agonists of the invention.Such cancers include, but are not limited to, malignant myeloma,leukemia and lymphoma.

Many of the pathologies associated with HIV are mediated by apoptosis,including HIV-induced nephropathy and HIV encephalitis. Thus, inadditional preferred embodiments, TNFR polynucleotides, polypeptides,and/or TNFR agonists of the invention are used to treat or prevent AIDSand pathologies associated with AIDS. Another embodiment of the presentinvention is directed to the use of TNFR-6 alpha and/or TNFR-6 beta(e.g., TR6-alpha- and/or TR6-beta-Fc or albumin fusion proteins) toreduce Fas ligand and/or AIM-II-mediated death of T cells inHIV-infected patients.

The state of Immunodificiency that defines AIDS is secondary to adecrease in the number and function of CD4⁺ T-lymphocytes. Recentreports estimate the daily loss of CD4⁺ T cells to be between 3.5×10⁷and 2×10⁹ cells (Wei X., et al., Nature 373:117-122 (1995)). One causeof CD4⁺ T cell depletion in the setting of HIV infection is believed tobe HIV-induced apoptosis (see, for example, Meyaard et al., Science257:217-219, (1992); Groux et al., J. Exp. Med, 175:331, (1992); andOyaizu et al., in Cell Activation and Apoptosis in HIV Infection,Andrieu and Lu, Eds., Plenum Press, New York, 1995, pp. 101-114).Indeed, HIV-induced apoptotic cell death has been demonstrated not onlyin vitro but also, more importantly, in infected individuals (Ameisen,J. C., AIDS 8:1197-1213 (1994); Finkel, T. H., and Banda, N. K., Curr.Opin. Immunol. 6:605-615(1995); Muro-Cacho, C. A. et al., J. Immunol.154:5555-5566 (1995)). Furthermore, apoptosis and CD4⁺ T-lymphocytedepletion is tightly correlated in different animal models of AIDS(Brunner, T., et al., Nature 373:441-444 (1995); Gougeon, M. L., et al.,AIDS Res. Hum. Retroviruses 9:553-563 (1993)) and, apoptosis is notobserved in those animal models in which viral replication does notresult in AIDS (Gougeon, M. L. et al., AIDS Res. Hum. Retroviruses9:553-563 (1993)). Further data indicates that uninfected but primed oractivated T lymphocytes from HIV-infected individuals undergo apoptosisafter encountering the Fas Ligand. Using monocytic cell lines thatresult in death following HIV infection, it has been demonstrated thatinfection of U937 cells with HIV results in the de novo expression ofFas ligand and that Fas ligand mediates HIV-induced apoptosis (Badley,A. D. et al., J. Virol. 70:199-206 (1996)). Further the TNF-familyligand was detectable in uninfected macrophages and its expression wasupregulated following HIV infection resulting in selective killing ofuninfected CD4 T-lymphocytes (Badley, A. D et al., J. Virol. 70:199-206(1996)). Further, additional studies have implicated Fas-mediatedapoptosis in loss of T cells in HIV individuals (Katsikis et al., J.Exp. Med. 181:2029-2036, 1995).

Thus, by the invention, a method for treating HIV⁺ individuals isprovided which involves administering TNFR and/or TNFR agonists of thepresent invention to reduce selective killing of CD4 T-lymphocytes.Modes of administration and dosages are discussed in detail below.

It is also possible that T cell apoptosis occurs through multiplemechanisms. Further at least some of the T cell death seen in HIVpatients may be mediated by AIM-II. While not wishing to be bound bytheory, such Fas ligand and/or AIM-II-mediated T cell death is believedto occur through the mechanism known as activation-induced cell death(AICD).

Activated human T cells are induced to undergo programmed cell death(apoptosis) upon triggering through the CD3/T cell receptor complex, aprocess termed activated-induced cell death (AICD). AICD of CD4 T cellsisolated from HIV-Infected asymptomatic individuals has been reported(Groux et al., supra). Thus, AICD may play a role in the depletion ofCD4+ T cells and the progression to AIDS in HIV-infected individuals.Thus, the present invention provides a method of inhibiting Fasligand-mediated and/or AIM-II-mediated T cell death in HIV patients,comprising administering a TNFR-6 alpha and/or TNFR-6 beta polypeptideof the invention to the patients. In one embodiment, the patient isasymptomatic when treatment with TNFR-6 alpha and/or TNFR-6 betacommences. If desired, prior to treatment, peripheral blood T cells maybe extracted from an HIV patient, and tested for susceptibility to Fasligand-mediated and/or AIM-II-mediated cell death by conventionalprocedures. In one embodiment, a patient's blood or plasma is contactedwith TNFR-6 alpha and/or TNFR-6 beta ex vivo. The TNFR-6 alpha and/orTNFR-6 beta may be bound to a suitable chromatography matrix known inthe art by conventional procedures. The patient's blood or plasma flowsthrough a chromatography column containing TNFR-6 alpha and/or TNFR-6beta polypeptides of the invention bound to the matrix, before beingreturned to the patient. The immobilized TNFR-6 alpha and/or TNFR-6 betabinds Fas ligand and/or AIM-II, thus removing Fas ligand and/or AIM-IIprotein from the patient's blood.

In additional embodiments a TNFR-6 alpha and/or TNFR-6 beta polypeptideof the invention may be administered in combination with otherinhibitors of T cell apoptosis. For example, at least some of the T celldeath seen in HIV patients is believed to be mediated by TRAIL(International application publication number WO 97/01633 herebyincorporated by reference). Thus, for example, a patient susceptible toboth Fas ligand mediated and TRAIL mediated T cell death may be treatedwith both an agent that blocks TRAIL/TRAIL-receptor interactions and anagent that blocks Fas-ligand/Fas interactions. Suitable agents that maybe administered with the polynucleotides and/or polypeptides of theinvention to block binding of TRAIL to TRAIL receptors include, but arenot limited to, soluble TRAIL receptor polypeptides (e.g., a solubleform of OPG, DR4 (International application publication number WO98/32856); TR5 (International application publication number WO98/30693); DR5 (International application publication number WO98/41629); TR10 (International application publication number WO98/54202)); multimeric forms of soluble TRAIL receptor polypeptides; andTRAIL receptor antibodies that bind the TRAIL receptor withouttransducing the biological signal that results in apoptosis, anti-TRAILantibodies that block binding of TRAIL to one or more TRAIL receptors,and muteins of TRAIL that bind TRAIL receptors but do not transduce thebiological signal that results in apoptosis. Preferably, the antibodiesemployed according to this method are monoclonal antibodies.

Suitable agents, which also block binding of Fas-ligand to Fas that maybe administered with the polynucleotides and polypeptides of the presentinvention include, but are not limited to, soluble Fas polypeptides;multimeric forms of soluble Fas polypeptides (e.g., dimers of sFas/Fc);anti-Fas antibodies that bind Fas without transducing the biologicalsignal that results in apoptosis; anti-Fas-ligand antibodies that blockbinding of Fas-ligand to Fas; and muteins of Fas-ligand that bind Fasbut do not transduce the biological signal that results in apoptosis.Examples of suitable agents for blocking Fas-L/Fas interactions,including blocking anti-Fas monoclonal antibodies, are described inInternational application publication number WO 95/10540, herebyincorporated by reference.

Suitable agents that may be administered with the polynucleotides and/orpolypeptides of the invention to block binding of AIM-II to AIM-IIreceptors include, but are not limited to, soluble AIM-II receptorpolypeptides (e.g., a soluble form of TR2 (International applicationpublication number WO 96/34095); LT beta receptor; and TR8(International application publication number WO 98/54201)); multimericforms of soluble AIM-II receptor polypeptides; and AIM-II receptorantibodies that bind the AIM-II receptor without transducing thebiological signal that results in apoptosis, anti-AIM-II antibodies thatblock binding of AIM-II to one or more AIM-II receptors, and muteins ofAIM-II that bind AIM-II receptors but do not transduce the biologicalsignal that results in apoptosis. Preferably, the antibodies employedaccording to this method are monoclonal antibodies.

In rejection of an allograft, the immune system of the recipient animalhas not previously been primed to respond because the immune system forthe most part is only primed by environmental antigens. Tissues fromother members of the same species have not been presented in the sameway that, for example, viruses and bacteria have been presented. In thecase of allograft rejection, immunosuppressive regimens are designed toprevent the immune system from reaching the effector stage. However, theimmune profile of xenograft rejection may resemble disease recurrencemore than allograft rejection. In the case of disease recurrence, theimmune system has already been activated, as evidenced by destruction ofthe native islet cells. Therefore, in disease recurrence the immunesystem is already at the effector stage. Antagonists of the presentinvention are able to suppress the immune response to both allograftsand xenografts because lymphocytes activated and differentiated intoeffector cells will express the TNFR polypeptide, and thereby aresusceptible to compounds which enhance TNFR activity. Thus, the presentinvention further provides a method for creating immune privilegedtissues. Antagonist of the invention can further be used in thetreatment of Inflammatory Bowel-Disease.

TNFR polynucleotides, polypeptides, and agonists of the invention mayalso be used to suppress immune responses. In one embodiment, the TNFRpolynucleotides, polypeptides, and agonists of the invention are used tominimize untoward effects associated with transplantation. In a specificembodiment, the TNFR polynucleotides, polypeptides, and agonists of theinvention are used to suppress Fas mediated immune responses (e.g., in amanner similar to an immunosuppressant such as, for example, rapamycinor cyclosporin). In another specific embodiment, the TNFRpolynucleotides, polypeptides, and agonists of the invention are used tosuppress AIM-II mediated immune responses.

Additionally, both graft rejection and graft vs. host disease are inpart triggered by apoptosis. Accordingly, an additional preferredembodiment, TNFR polynucleotides, polypeptides, and/or TNFR agonists ofthe invention are used to treat and prevent and/or reduce graftrejection. In a further preferred embodiment, TNFR polynucleotides,polypeptides, and/or TNFR agonists of the invention are used to treatand prevent and/or reduce graft vs. host disease.

Additionally, TNFR-6 alpha and/or TNFR-6 beta polypeptides,polynucleotides, and/or agonists may be used to treat or prevent graftrejection (e.g., xenograft and allograft rejection (e.g, acute allograftrejection)) and/or medical conditions associated with graft rejection.In a specific embodiment, TNFR-6 alpha and/or TNFR-6 beta polypeptides,polynucleotides, and/or agonists of the invention are used to treat orprevent acute allograft rejection and/or medical conditions associatedwith acute allograft rejection. In a further specific embodiment, TNFR-6alpha and/or TNFR-6 beta polypeptides, polynucleotides, and/or agonistsof the invention are used to treat or prevent acute allograft rejectionof a kidney and/or medical conditions associated with acute allograftrejection of a kidney.

Fas ligand is a type II membrane protein that induces apoptosis bybinding to Fas. Fas ligand is expressed in activated T cells, and worksas an effector of cytotoxic lymphocytes. Molecular and genetic analysisof Fas and Fas ligand have indicated that mouse lymphoproliferationmutation (lpr) and generalized lymphoproliferative disease (gld) aremutations of Fas and Fas ligand respectively. The lpr of gld micedevelop lymphadenopathy, and suffer from autoimmune disease. Based onthese phenotypes and other studies, it is believed that the Fas systemis involved in the apoptotic process during T-cell development,specifically peripheral clonal deletion or activation-induced suicide ofmature T cells. In addition to the activated lymphocytes, Fas isexpressed in the liver, heart and lung. Administration of agonisticanti-Fas antibody into mice has been shown to induce apoptosis in theliver and to quickly kill the mice, causing liver damage. These findingsindicate that the Fas system plays a role not only in the physiologicalprocess of lymphocyte development, but also in the cytotoxicT-lymphocyte-mediated disease such as fulminant hepatitis and/orhepatitis resulting from viral infection or toxic agents. As discussedherein, TNFR-6 alpha and/or TNFR-6 beta binds Fas ligand, and thusfunctions as an antagonist of Fas-ligand mediated activity. Accordingly,the TNFR-6 alpha and/or TNFR-6 beta polypeptides and/or polynucleotidesof the invention, and/or agonists thereof, may be used to treat orprevent lymphoproliferative disorders (e.g., lymphadenopathy and othersdescribed herein), autoimmune disorders (e.g., autoimmune diabetes,systemic lupus erythematosus, Grave's disease, Hashimoto's thyroiditis,immune-related glomerulonephritis, autoimmune gastritis, autoimmunethrombocytopenic purpura, multiple sclerosis, rheumatoid arthritis, andothers described herein), and/or liver disease (e.g., acute and chronichepatitis, and cirrhosis).

In a specific embodimen, TNFR polynucleotides, polypeptides, and/oragonists of the invention are used to treat or prevent hepatitis and/ortissue/cell damage or destruction and/or medical conditions associatedwith hepatitis. In a specific embodiment TNFR polynucleotides,polypeptides, and/or agonists of the invention are used to treat orprevent fulminant hepatitis and/or medical conditions associated withfulminant hepatitis.

In a specific embodiment, TNFR polynucleotides, polypeptides, and/oragonists of the invention are used to treat or prevent systemic lupuserythematosus (SLE) and/or tissue/cell damage or destruction and/ormedical conditions associated with SLE. In a further specificembodiment, TNFR polynucleotides, polypeptides, and/or agonists of theinvention are used to treat or prevent skin lesions in SLE patients.

In a specific embodiment, TNFR polynucleotides, polypeptides, and/oragonists of the invention are used to treat or prevent insulin-dependentdiabetes mellitus and/or tissue/cell damage or destruction and/ormedical conditions associated with insulin-dependent diabetes mellitus.In a further specific embodiment, TNFR polynucleotides, polypeptides,and/or agonists of the invention are prior to, during, or immediatelyafter the onset of diabetes to reduce or prevent damage to islet cellsand/or to reduce exogenous insulin requirement.

In a specific embodiment TNFR polynucleotides, polypeptides, and/oragonists of the invention are used to treat or prevent toxic epidermalnecrolysis (TEN) and/or tissue/cell damage or destruction, and/ormedical conditions associated with TEN. In a further specificembodiment, TNFR polynucleotides, polypeptides, and/or agonists of theinvention are used to treat or prevent Lyell's syndrome.

Hepatitis virus (e.g., Hepatitis B virus and Hepatitis C virus) is amajor causative agent of chronic liver disease. In Hepatitis infection,Fas expression in hepatocytes is up-regulated in accordance with theseverity of liver inflammation. When Hepatitis virus-specific T cellsmigrate into hepatocytes and recognize the viral antigen via the T cellreceptor, they become activated and express Fas ligand that cantransduce the apoptotic death signal to Fas-bearing hepatocytes. Thus,the Fas system plays an important role in liver cell injury by viralhepatitis. Accordingly, in specific embodiments, the TNFR-6 alpha and/orTNFR-6 beta polypeptides and/or polynucleotides of the invention and/oragonists or antagonists thereof, are used to treat or prevent hepatitisresulting from viral infection (e.g., infection resulting form HepatitisB virus or Hepatitis C virus infection). In one embodiment, a patient'sblood or plasma is contacted with TNFR-6 alpha and/or TNFR-6 betapolypeptides of the invention ex vivo. The TNFR-6 alpha and/or TNFR-6beta may be bound to a suitable chromatography matrix by conventionalprocedures. According to this embodiment, the patient's blood or plasmaflows through a chromatography column containing TNFR-6 alpha and/orTNFR-6 beta bound to the matrix, before being returned to the patient.The immobilized TNFR-6 alpha and/or TNFR-6 beta binds Fas-ligand, thusremoving Fas-ligand protein from the patient's blood.

In a specific embodiment, TNFR-6 alpha and/or TNFR-6 beta polypeptides,polynucleotides, and/or agonists or antagonists of the invention may beused to treat or prevent renal failure (e.g., chronic renal failure),and/or tissue/cell damage or destruction (e.g., tubular epithelial celldeletion) and/or medical conditions associated with renal failure.

In a specific embodiment, TNFR-6 alpha and/or TNFR-6 beta polypeptides,polynucleotides, and/or agonists or antagonists of the invention may beused to regulate (i.e., stimulate or inhibit) bone growth. In specificembodiments TNFR-6 alpha and/or TNFR-6 beta polypeptides,polynucleotides, and/or agonists or antagonists of the invention areused to stimulate bone growth. Specific diseases or conditions that maybe treated or prevented with the compositions of the invention include,but are not limited to, bone fractures, and defects, and disorders whichresult in weakened bones such as osteoporosis, osteomalacia, andage-related loss of bone mass.

TNFR-6 alpha and/or TNFR-6 beta polypeptides or polynucleotides encodingTNFR-6 alpha and/or TNFR-6 beta of the invention, and/or agonists orantagonists thereof may be used to treat or prevent cardiovasculardisorders, including peripheral artery disease, such as limb ischemia.

Cardiovascular disorders include cardiovascular abnormalities, such asarterio-arterial fistula, arteriovenous fistula, cerebral arteriovenousmalformations, congenital heart defects, pulmonary atresia, and ScimitarSyndrome. Congenital heart defects include aortic coarctation, cortriatriatum, coronary vessel anomalies, crisscross heart, dextrocardia,patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex,hypoplastic left heart syndrome, levocardia, tetralogy of fallot,transposition of great vessels, double outlet right ventricle, tricuspidatresia, persistent truncus arteriosus, and heart septal defects, suchas aortopulmonary septal defect, endocardial cushion defects,Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septaldefects.

Cardiovascular disorders also include heart disease, such asatherosclerosis, arrhythmias, carcinoid heart disease, high cardiacoutput, low cardiac output, cardiac tamponade, endocarditis (includingbacterial), heart aneurysm, cardiac arrest, congestive heart failure(e.g., chronic congestive heart failure), congestive cardiomyopathy,paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestivecardiomyopathy, left ventricular hypertrophy, right ventricularhypertrophy, post-infarction heart rupture, ventricular septal rupture,heart valve diseases, myocardial diseases, myocardial ischemia,pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryfibrosis, pulmonary heart disease, rheumatic heart disease, ventriculardysfunction, hyperemia, cardiovascular pregnancy complications, ScimitarSyndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

In a specific embodiment, TNFR-6 alpha and/or TNFR-6 betapolynucleotides, polypeptides, or agonists of the invention may be usedto treat and/or prevent chronic congestive heart failure and/or medicalconditions associated chronic congestive heart failure.

In another specific embodiment, TNFR-6 alpha and/or TNFR-6 betapolynucleotides, polypeptides, or agonists of the invention may be usedto treat and/or prevent pulmonary injury or disease (e.g., pulmonaryfibrosis and chronic obstructive pulmonary diseases, such as, forexample, emphysema and chronic bronchitis), and/or tissue/cell damage ordestruction (e.g., alveolar wall and/or bronchiolar wall destruction)and/or medical conditions associated with pulmonary injury or disease.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include carotid artery diseases, cerebralamyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebralarteriosclerosis, cerebral arteriovenous malformation, cerebral arterydiseases, cerebral embolism and thrombosis, carotid artery thrombosis,sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epiduralhematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebralinfarction, cerebral ischemia (including transient), subclavian stealsyndrome, periventricular leukomalacia, vascular headache, clusterheadache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromoboembolisms. Thromboses include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

In one embodiment, TNFR-6 alpha and/or TNFR-6 beta polypeptides,polynucleotides and/or agonists or antagonists of the invention are usedto treat or prevent thrombotic microangiopathies. One such disorder isthrombotic thrombocytopenic purpura (TTP) (Kwaan, H. C., Semin. Hematol.24:71 (1987); Thompson et al., Blood 80:1890 (1992)). IncreasingTTP-associated mortality rates have been reported by the U.S. Centersfor Disease Control (Torok et al., Am. J. Hematol. 50:84 (1995)). Plasmafrom patients afflicted with TTP (including HIV+ and HIV-patients)induces apoptosis of human endothelial cells of dermal microvascularorigin, but not large vessel origin (Laurence et al., Blood 87:3245(1996)). Plasma of TTP patients thus is thought to contain one or morefactors that directly or indirectly induce apoptosis. An anti-Fasblocking antibody has been shown to reduce TTP plasma-mediated apoptosisof microvascular endothelial cells (Lawrence et al., Blood 87:3245(1996); hereby incorporated by reference). Accordingly, Fas ligandpresent in the serum of TTP patients is likely to play a role ininducing apoptosis of microvascular endothelial cells. Anotherthrombotic microangiopathy is hemolytic-uremic syndrome (HUS) (Moake, J.L., Lancet, 343:393, (1994); Melnyk et al., (Arch. Intern. Med,155:2077, (1995); Thompson et al., supra). Thus, in one embodiment, theinvention is directed to use of TNFR-6 alpha and/or TNFR-6 beta to treator prevent the condition that is often referred to as “adult HUS” (eventhough it can strike children as well). A disorder known aschildhood/diarrhea-associated HUS differs in etiology from adult HUS. Inanother embodiment, conditions characterized by clotting of small bloodvessels may be treated using TNFR-6 alpha and/or TNFR-6 betapolypeptides and/or polynucleotides of the invention. Such conditionsinclude, but are not limited to, those described herein. For example,cardiac problems seen in about 5-10% of pediatric AIDS patients arebelieved to involve clotting of small blood vessels. Breakdown of themicrovasculature in the heart has been reported in multiple sclerosispatients. As a further example, treatment of systemic lupuserythematosus (SLE) is contemplated. In one embodiment, a patient'sblood or plasma is contacted with TNFR-6 alpha and/or TNFR-6 betapolypeptides of the invention ex vivo. The TNFR-6 alpha and/or TNFR-6beta may be bound to a suitable chromatography matrix using techniquesknown in the art. According to this embodiment, the patient's blood orplasma flows through a chromatography column containing TNFR-6 alphaand/or TNFR-6 beta bound to the matrix, before being returned to thepatient. The immobilized TNFR-6 alpha and/or TNFR-6 beta binds Fasligand and/or AIM-II, thus removing Fas ligand protein from thepatient's blood. Alternatively, TNFR-6 alpha and/or TNFR-6 beta may beadministered in vivo to a patient afflicted with a thromboticmicroangiopathy. In one embodiment, a TNFR-6 alpha and/or TNFR-6 betapolynucleotide or polypeptide of the invention is administered to thepatient. Thus, the present invention provides a method for treating athrombotic microangiopathy, involving use of an effective amount of aTNFR-6 alpha and/or TNFR-6 beta polypeptide of the invention. A TNFR-6alpha and/or TNFR-6 beta polypeptide may be employed in in vivo or exvivo procedures, to inhibit Fas ligand-mediated and/or AIM-II-mediateddamage to (e.g., apoptosis of) microvascular endothelial cells.

TNFR-6 alpha and/or TNFR-6 beta polypeptides and polynucleodies of theinvention may be employed in conjunction with other agents useful intreating a particular disorder. For example, in an in vitro studyreported by Laurence et al. (Blood 87:3245, 1996), some reduction of TTPplasma-mediated apoptosis of microvascular endothelial cells wasachieved by using an anti-Fas blocking antibody, aurintricarboxylicacid, or normal plasma depleted of cryoprecipitate. Thus, a patient maybe treated in combination with an additional agent that inhibitsFas-ligand-mediated apoptosis of endothelial cells such as, for example,an agent described above. In one embodiment, TNFR-6 alpha and/or TNFR-6beta polypeptides of the invention and an anti-FAS blocking antibody areadministered to a patient afflicted with a disorder characterized bythrombotic microanglopathy, such as TTP or HUS. Examples of blockingmonoclonal antibodies directed against Fas antigen (CD95) are describedin International Application publication number WO 95/10540, herebyincorporated by reference.

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

The present invention provides for treatment of diseases or disordersassociated with neovascularization by administration of the TNFR-6 alphaand/or TNFR-6 beta polynucleotides and/or polypeptides of the invention.Malignant and metastatic conditions which can be treated with thepolynucleotides and polypeptides of the invention include, but are notlimited to, malignancies, solid tumors, and cancers described herein andotherwise known in the art (for a review of such disorders, see Fishmanet al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)):

Ocular disorders associated with neovascularization which can be treatedwith the TNFR-6 alpha and/or TNFR-6 beta polynucleotides andpolypeptides of the present invention (including TNFR agonists and/orantagonists) include, but are not limited to: neovascular glaucoma,diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis,retinopathy of prematurity macular degeneration, corneal graftneovascularization, as well as other eye inflammatory diseases, oculartumors and diseases associated with choroidal or irisneovascularization. See, e.g., reviews by Waltman et al., Am. J.Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312(1978).

In another embodiment, TNFR-6 alpha and/or TNFR-6 beta polypeptides,polynucleotides and/or agonists or antagonists of the invention are usedto stimulate differentiation and/or survival of photoreceptor cellsand/or to treat or prevent diseases, disorders, or conditions associatedwith decreased number, differentiation and/or survival of photoreceptorcells.

Additionally, disorders which can be treated with the TNFR-6 alphaand/or TNFR-6 beta polynucleotides and polypeptides of the presentinvention (including TNFR agonist and/or antagonists) include, but arenot limited to, hemangioma, arthritis, psoriasis, angiofibroma,atherosclerotic plaques, delayed wound healing, granulations, hemophilicjoints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome,pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

In additional embodiments, TNFR-6 alpha and/or TNFR-6 betapolynucleotides, polynucleotides and/or other compositions of theinvention (e.g., TNFR-6 alpha and/or TNFR-6 beta Fc- or albumin-fusionproteins or anti-TNFR-6 alpha and/or anti-TNFR-6 beta antibodies) areused to treat or prevent diseases or conditions associated with allergyand/or inflammation.

As demonstrated in Example 24 below, it has been shown that TR6-alphaand TR6-beta interact with TNF-gamma-beta, a TNF ligand family memberdescribed in detail in International Publication Numbers WO96/14328,WO00/66608, and WO00/08139. TNF-gamma-beta is a proinflammatory moleculeas evidenced by its ability to induce T cell proliferation and secretionof Interferon-gamma and GM-CSF by T cells. TNF-gamma-beta is also ableto enhance an in vivo mixed lymphocyte reaction (MLR) as measured by theparent-into-F1 model of acute graft vs. host disease in which C57BL/6splenic T cells are transferred into (BALB/c×C57BL/6) F1 mice. Thus, theability of TNFR-6 alpha to bind TNF-gamma-beta and to preventTNF-gamma-beta induced activities (see Example 24) suggests that TNFR-6alpha and or TNF-6beta polynucleotides and polypeptides are useful asinhibitors of TNF-gamma-beta function.

In specific embodiments, TNFR-6 alpha and/or TNFR-6 beta polynucleotidesand polypeptides and fragments or variants thereof (e.g. soluble formsof TNFR-6 alpha such as TNFR-6 alpha Fc fusion proteins or TNFR-6 alphaalbumin fusion proteins) are useful for the prevention, diagnosis andtreatment of inflammation and/or inflammatory diseases and disorders. Inparticular embodiments, the present invention provides a method ofdiagnosing, diagnosing, treating, preventing or amelioratinginflammatory diseases or disorders comprising or alternativelyconsisting of, administering to an animal, preferably a human, in whichsuch treatment, prevention or amelioration is desired, a TNFR-6 alphaand/or TNFR-6 beta polynucleotide or polypeptide or a fragment orvariant thereof (e.g. soluble forms of TNFR-6 alpha and/or TNFR-6 betasuch as a TNFR-6 alpha and/or TNFR-6 beta Fc- or albumin-fusion protein)in an amount effective to treat prevent or ameliorate the inflammatorydisease or disorder. In specific embodiments, the inflammatory diseaseor disorder is inflammatory bowel disease. In specific embodiments, theinflammatory disease or disorder is encephalitis. In specificembodiments, the inflammatory disease or disorder is atherosclerosis. Inspecific embodiments, the inflammatory disease or disorder is psoriasis.The present invention further provides compositions comprising theTNFR-6 alpha and/or TNFR-6 beta polynucleotide or polypeptide or afragment or variant thereof (e.g. soluble forms of TNFR-6 alpha and/orTNFR-6 beta such as a TNFR-6 alpha and/or TNFR-6 beta Fc- oralbumin-fusion protein) and a carrier for use in the above-describedmethod of diagnosing, treating, preventing or ameliorating inflammatorydiseases and disorders.

In specific embodiments, the present invention provides a method ofdiagnosing, treating, preventing or ameliorating inflammation comprisingor alternatively consisting of, administering to an animal, preferably ahuman, in which such treatment, prevention or amelioration is desired, aTNFR-6 alpha and/or TNFR-6 beta polynucleotide or polypeptide or afragment or variant thereof (e.g. soluble forms of TNFR-6 alpha and/orTNFR-6 beta such as a TNFR-6 alpha and/or TNFR-6 beta Fc- oralbumin-fusion protein) in an amount effective to treat prevent orameliorate the inflammation. The present invention further providescompositions comprising a TNFR-6 alpha and/or TNFR-6 beta polynucleotideor polypeptide or a fragment or variant thereof (e.g. soluble forms ofTNFR-6 alpha and/or TNFR-6 beta such as a TNFR-6 alpha and/or TNFR-6beta Fc- or albumin-fusion protein) and a carrier for use in theabove-described method of diagnosing, treating, preventing orameliorating inflammation.

In specific embodiments, the present invention provides a method ofdiagnosing, treating, preventing or ameliorating graft versus hostdisease (GVHD) comprising or alternatively consisting of, administeringto an animal, preferably a human, in which such treatment, prevention oramelioration is desired, a TNFR-6 alpha and/or TNFR-6 betapolynucleotide or polypeptide or a fragment or variant thereof (e.g.soluble forms of TNFR-6 alpha and/or TNFR-6 beta such as a TNFR-6 alphaand/or TNFR-6 beta Fc- or albumin-fusion protein) in an amount effectiveto treat prevent or ameliorate the GVHD. The present invention furtherprovides compositions comprising a TNFR-6 alpha and/or TNFR-6 betapolynucleotide or polypeptide or a fragment or variant thereof (e.g.soluble forms of TNFR-6 alpha and/or TNFR-6 beta such as a TNFR-6 alphaand/or TNFR-6 beta Fc- or albumin-fusion protein) and a carrier for usein the above-described method of diagnosing, treating, preventing orameliorating GVHD.

In other embodiments, the present invention provides a method ofdiagnosing, treating, preventing or ameliorating autoimmune diseases anddisorders comprising or alternatively consisting of, administering to ananimal, preferably a human, in which such treatment, prevention oramelioration is desired, a TNFR-6 alpha and/or TNFR-6 betapolynucleotide or polypeptide or a fragment or variant thereof (e.g.soluble forms of TNFR-6 alpha and/or TNFR-6 beta such as a TNFR-6 alphaand/or TNFR-6 beta Fc- or albumin-fusion protein) in an amount effectiveto treat prevent or ameliorate the autoimmune disease or disorder. Inspecific embodiments, the autoimmune disease or disorder is systemiclupus erythematosus. In specific embodiments, the autoimmune disease ordisorder is arthritis, particularly rheumatoid arthritis. In specificembodiments, the autoimmune disease or disorder is multiple sclerosis.In specific embodiments, the autoimmune disease or disorder is Crohn'sdisease. In specific embodiments, the autoimmune disease or disorder isautoimmune encephalitis. The present invention further providescompositions comprising a TNFR-6 alpha and/or TNFR-6 beta polynucleotideor polypeptide or a fragment or variant thereof (e.g. soluble forms ofTNFR-6 alpha and/or TNFR-6 beta such as a TNFR-6 alpha and/or TNFR-6beta Fc- or albumin-fusion protein) and a carrier for use in theabove-described method of diagnosing, treating, preventing orameliorating autoimmune diseases and disorders.

In specific embodiments, the present invention provides a method ofdiagnosing, treating, preventing or ameliorating allergy or asthmacomprising or alternatively consisting of, administering to an animal,preferably a human, in which such treatment, prevention or ameliorationis desired, a TNFR-6 alpha and/or TNFR-6 beta polynucleotide orpolypeptide or a fragment or variant thereof (e.g. soluble forms ofTNFR-6 alpha and/or TNFR-6 beta such as a TNFR-6 alpha and/or TNFR-6beta Fc- or albumin-fusion protein) or fragment or variant thereof in anamount effective to treat prevent or ameliorate the allergy or asthma.The present invention further provides compositions comprising a TNFR-6alpha and/or TNFR-6 beta polynucleotide or polypeptide or a fragment orvariant thereof (e.g. soluble forms of TNFR-6 alpha and/or TNFR-6 betasuch as a TNFR-6 alpha and/or TNFR-6 beta Fc- or albumin-fusion protein)and a carrier for use in the above-described method of diagnosing,treating, preventing or ameliorating allergy or asthma.

The present invention further encompasses methods and compositions forreducing T cell activation, comprising, or alternatively consisting of,contacting an effective amount of a TNFR-6 alpha and/or TNFR-6 betapolypeptide or a fragment or variant thereof (e.g. soluble forms ofTNFR-6 alpha and/or TNFR-6 beta such as a TNFR-6 alpha and/or TNFR-6beta Fc- or albumin-fusion protein) with cells of hematopoietic origin,wherein the effective amount of the TNFR-6 alpha and/or TNFR-6 betapolypeptide or a fragment or variant thereof (e.g. soluble forms ofTNFR-6 alpha and/or TNFR-6 beta such as a TNFR-6 alpha and/or TNFR-6beta Fc- or albumin-fusion protein) reduces T cell activation. Inpreferred embodiments, the cells of hematopoietic origin are T cells. Inother preferred embodiments, the effective amount of a TNFR-6 alphaand/or TNFR-6 beta polypeptide or a fragment or variant thereof (e.g.soluble forms of TNFR-6 alpha and/or TNFR-6 beta such as a TNFR-6 alphaand/or TNFR-6 beta Fc- or albumin-fusion protein) reducesTNF-gamma-alpha and/or TNF-gamma beta induced T cell activation.

The present invention further encompasses methods and compositions forreducing T cell activation comprising, or alternatively consisting of,administering to an animal, preferably a human, in which such reductionis desired, a TNFR-6 alpha and/or TNFR-6 beta polynucleotide orpolypeptide or a fragment or variant thereof (e.g. soluble forms ofTNFR-6 alpha and/or TNFR-6 beta such as a TNFR-6 alpha and/or TNFR-6beta Fc- or albumin-fusion protein) or fragment or variant thereof in anamount effective to reduce T cell activation. The present inventionfurther provides compositions comprising a TNFR-6 alpha and/or TNFR-6beta polynucleotide or polypeptide or a fragment or variant thereof(e.g. soluble forms of TNFR-6 alpha and/or TNFR-6 beta such as a TNFR-6alpha and/or TNFR-6 beta Fc- or albumin-fusion protein) and a carrierfor use in the above-described method of reducing T cell activation.

In a specific embodiment TNFR polynucleotides, polypeptides and/oragonists or antagonists thereof may be used to treat or preventthyroid-associated opthalmopathy and/or tissue/cell damage ordestruction, and/or medical conditions associated withthyroid-associated opthalmopathy.

In a specific embodiment, TNFR polynucleotides, polypeptides, oragonists of the invention are used to prolong protein expression aftergene therapy by inhibiting or reducing elimination of transgeneexpressing cells.

In further embodiments, the TNFR-6 alpha and/or TNFR-6 betapolynucleotides and/or polynucleotides, and/or agonists or antagoniststhereof, are used to promote wound healing.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are useful in the diagnosis and treatment orprevention of a wide range of diseases and/or conditions. Such diseasesand conditions include, but are not limited to, cancer (e.g., immunecell related cancers, breast cancer, prostate cancer, ovarian cancer,follicular lymphoma, cancer associated with mutation or alteration ofp53, brain tumor, bladder cancer, uterocervical cancer, colon cancer,colorectal cancer, non-small cell carcinoma of the lung, small cellcarcinoma of the lung, stomach cancer, etc.), lymphoproliferativedisorders (e.g., lymphadenopathy), microbial (e.g., viral, bacterial,etc.) infection (e.g., HIV-1 infection, HIV-2 infection, herpesvirusinfection (including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6,HHV-7, EBV), adenovirus infection, poxvirus infection, human papillomavirus infection, hepatitis infection (e.g., HAV, HBV, HCV, etc.),Helicobacter pylori infection, invasive Staphylococcia, etc.), parasiticinfection, nephritis, bone disease (e.g., osteoporosis),atherosclerosis, pain, cardiovascular disorders (e.g.,neovascularization, hypovascularization or reduced circulation (e.g.,ischemic disease (e.g., myocardial infarction, stroke, etc.))), AIDS,allergy, inflammation, neurodegenerative disease (e.g., Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, pigmentaryretinitis, cerebellar degeneration, etc.), graft rejection (acute andchronic), graft vs. host disease, diseases due to osteomyelodysplasia(e.g., aplastic anemia, etc.), joint tissue destruction in rheumatism,liver disease (e.g., acute and chronic hepatitis, liver injury, andcirrhosis), autoimmune disease (e.g., multiple sclerosis, rheumatoidarthritis, systemic lupus erythematosus, immune complexglomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenicpurpura, Grave's disease, Hashimoto's thyroiditis, etc.), cardiomyopathy(e.g., dilated cardiomyopathy), diabetes, diabetic complications (e.g.,diabetic nephropathy, diabetic neuropathy, diabetic retinopathy),influenza, asthma, psoriasis, glomerulonephritis, septic shock, andulcerative colitis.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are useful in promoting angiogenesis,regulating hematopoiesis and wound healing (e.g., wounds, burns, andbone fractures).

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are also useful as an adjuvant to enhanceimmune responsiveness to specific antigen, anti-viral immune responses.

More generally, polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof are useful in regulating(i.e., elevating or reducing) immune response. For example,polynucleotides and/or polypeptides of the invention may be useful inpreparation or recovery from surgery, trauma, radiation therapy,chemotherapy, and transplantation, or may be used to boost immuneresponse and/or recovery in the elderly and immunocompromisedindividuals. Alternatively, polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof are useful asimmunosuppressive agents, for example in the treatment or prevention ofautoimmune disorders. In specific embodiments, polynucleotides and/orpolypeptides of the invention are used to treat or prevent chronicinflammatory, allergic or autoimmune conditions, such as those describedherein or are otherwise known in the art.

In one aspect, the present invention is directed to a method forenhancing apoptosis induced by a TNF-family ligand, which involvesadministering to a patient (preferably a human) a TNFR antagonists(e.g., an anti-TNFR antibody or TNFR polypeptide fragment). Preferably,the TNFR antagonist is administered to treat a disease or conditionwherein increased cell survival is exhibited. Antagonists of theinvention include soluble forms of TNFR and monoclonal antibodiesdirected against the TNFR polypeptide.

By “antagonist” is intended naturally occurring and synthetic compoundscapable of enhancing or potentiating apoptosis. By “agonist” is intendednaturally occurring and synthetic compounds capable of inhibitingapoptosis. Whether any candidate “agonist” or “antagonist” of thepresent invention can inhibit or enhance apoptosis can be determinedusing art-known TNF-family ligand/receptor cellular response assays,including those described in more detail below.

One such screening procedure involves the use of melanophores which aretransfected to express the receptor of the present invention. Such ascreening technique is described in International applicationpublication number WO 92/01810, published Feb. 6, 1992. Such an assaymay be employed, for example, for screening for a compound whichinhibits (or enhances) activation of the receptor polypeptide of thepresent invention by contacting the melanophore cells which encode thereceptor with both a TNF-family ligand and the candidate antagonist (oragonist). Inhibition or enhancement of the signal generated by theligand indicates that the compound is an antagonist or agonist of theligand/receptor signaling pathway.

Other screening techniques include the use of cells which express thereceptor (for example, transfected CHO cells) in a system which measuresextracellular pH changes caused by receptor activation, for example, asdescribed in Science 246:181-296 (October 1989). For example, compoundsmay be contacted with a cell which expresses the receptor polypeptide ofthe present invention and a second messenger response, e.g., signaltransduction or pH changes, may be measured to determine whether thepotential compound activates or inhibits the receptor.

Another such screening technique involves introducing RNA encoding thereceptor into Xenopus oocytes to transiently express the receptor. Thereceptor oocytes may then be contacted with the receptor ligand and acompound to be screened, followed by detection of inhibition oractivation of a calcium signal in the case of screening for compoundswhich are thought to inhibit activation of the receptor.

Another screening technique involves expressing in cells a constructwherein the receptor is linked to a phospholipase C or D. Such cellsinclude endothelial cells, smooth muscle cells, embryonic kidney cells,etc. The screening may be accomplished as hereinabove described bydetecting activation of the receptor or inhibition of activation of thereceptor from the phospholipase signal.

Another method involves screening for compounds which inhibit activationof the receptor polypeptide of the present invention antagonists bydetermining inhibition of binding of labeled ligand to cells which havethe receptor on the surface thereof. Such a method involves transfectinga eukaryotic cell with DNA encoding the receptor such that the cellexpresses the receptor on its surface and contacting the cell with acompound in the presence of a labeled form of a known ligand. The ligandcan be labeled, e.g., by radioactivity. The amount of labeled ligandbound to the receptors is measured, e.g., by measuring radioactivity ofthe receptors. If the compound binds to the receptor as determined by areduction of labeled ligand which binds to the receptors, the binding oflabeled ligand to the receptor is inhibited.

Further screening assays for agonist and antagonist of the presentinvention are described in Tartaglia, L. A., and Goeddel, D. V., J.Biol. Chem. 267(7):43044307(1992).

Thus, in a further aspect, a screening method is provided fordetermining whether a candidate agonist or antagonist is capable ofenhancing or inhibiting a cellular response to a TNF-family ligand. Themethod involves contacting cells which express the TNFR polypeptide witha candidate compound and a TNF-family ligand, assaying a cellularresponse, and comparing the cellular response to a standard cellularresponse, the standard being assayed when contact is made with theligand in absence of the candidate compound, whereby an increasedcellular response over the standard indicates that the candidatecompound is an agonist of the ligand/receptor signaling pathway and adecreased cellular response compared to the standard indicates that thecandidate compound is an antagonist of the ligand/receptor signalingpathway. By “assaying a cellular response” is intended qualitatively orquantitatively measuring a cellular response to a candidate compoundand/or a TNF-family ligand (e.g., determining or estimating an increaseor decrease in T cell proliferation or tritiated thymidine labeling). Bythe invention, a cell expressing the TNFR polypeptide can be contactedwith either an endogenous or exogenously administered TNF-family ligand.

Agonist according to the present invention include naturally occurringand synthetic compounds such as, for example, TNF family ligand peptidefragments, transforming growth factor, neurotransmitters (such asglutamate, dopamine, N-methyl-D-aspartate), tumor suppressors (p53),cytolytic T cells and antimetabolites. Preferred agonists includechemotherapeutic drugs such as, for example, cisplatin, doxorubicin,bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate andvincristine. Others include ethanol and -amyloid peptide. (Science267:1457-1458 (1995)). Further preferred agonists include polyclonal andmonoclonal antibodies raised against the TNFR polypeptide, or a fragmentthereof. Such agonist antibodies raised against a TNF-family receptorare disclosed in Tartaglia, L. A., et al., Proc. Natl. Acad. Sci. USA88:9292-9296 (1991); and Tartaglia, L. A., and Goeddel, D. V., J. Biol.Chem. 267 (7):4304-4307 (1992) See, also, International applicationpublication number WO 94/09137.

Antagonists according to the present invention include naturallyoccurring and synthetic compounds such as, for example, the CD40 ligand,neutral amino acids, zinc, estrogen, androgens, viral genes (such asAdenovirus EIB, Baculovirus p35 and IAP, Cowpox virus crmA, Epstein-Barrvirus BHRF1, LMP-1, African swine fever virus LMW5-HL, and Herpesvirusyl 34.5), calpain inhibitors, cysteine protease inhibitors, and tumorpromoters (such as PMA, Phenobarbital, and -Hexachlorocyclohexane).Other antagonists include polyclonal and monoclonal antagonistantibodies raised against the TNFR polypeptides or a fragment thereof.Such antagonist antibodies raised against a TNF-family receptor aredescribed in Tartaglia, L. A., and Goeddel, D. V., J. Biol. Chem.267(7):43044307 (1992) and Tartaglia, L. A. et al., Cell 73:213-216(1993). See, also, International application publication number WO94/09137.

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in TNFR, orthe complementary strand thereof, and/or to nucleotide sequencescontained in the deposited clones (ATCC Deposit Nos. 97810 and 97809).In one embodiment, antisense sequence is generated internally by theorganism, in another embodiment, the antisense sequence is separatelyadministered (see, for example, O'Connor, J., Neurochem. 56:560 (1991)and Oligodeoxynucleotides as Anitsense Inhibitors of Gene Expression,CRC Press, Boca Raton, Fla. (1988). Antisense technology can be used tocontrol gene expression through antisense DNA or RNA, or throughtriple-helix formation. Antisense techniques are discussed for example,in Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Triple helix formation is discussed in, for instance, Lee etal., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science241:456 (1988); and Dervan et al., Science 251:1300(1991). The methodsare based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the TNFR antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the TNFR antisense nucleic acid. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others know inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding TNFR, or fragments thereof, can beby any promoter known in the art to act in vertebrate, preferably humancells. Such promoters can be inducible or constitutive. Such promotersinclude, but are not limited to, the SV40 early promoter region(Bernoist and Chambon, Nature 29:304-310 (1981), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al.,Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner et al.,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatorysequences of the metallothionein gene (Brinster, et al., Nature296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a TNFR gene.However, absolute complementarity, although preferred, is not required.A sequence “complementary to at least a portion of an RNA,” referred toherein, means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case of doublestranded TNFR antisense nucleic acids, a single strand of the duplex DNAmay thus be tested, or triplex formation may be assayed. The ability tohybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid Generally, the larger thehybridizing nucleic acid, the more base mismatches with a TNFR RNA itmay contain and still form a stable duplex (or triplex as the case maybe). One skilled in the art can ascertain a tolerable degree of mismatchby use of standard procedures to determine the melting point of thehybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′-non-translated, non-coding regions of the TNFR shown in FIGS.1 and 2A-B could be used in an antisense approach to inhibit translationof endogenous TNFR mRNA. Oligonucleotides complementary to the 5′untranslated region of the mRNA should include the complement of the AUGstart codon. Antisense oligonucleotides complementary to mRNA codingregions are less efficient inhibitors of translation but could be usedin accordance with the invention. Whether designed to hybridize to the5′-, 3′- or coding region of TNFR mRNA, antisense nucleic acids shouldbe at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides or at least 50nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad Sci. U.S.A.86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad Sci. 84:648-652(1987); PCT Publication No. WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., BioTechniques 6:958-976 (1988)) or intercalatingagents. (See, e.g., Zon, Pharm. Res. 5:539-549 (1988)). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res. 15:6625-6641 (1987)). The oligonucleotide isa2¢-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res. 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. USA.85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the TNFR coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., International application publicationnumber WO 90/11364, published Oct. 4, 1990; Sarver et al, Science247:1222-1225 (1990). While ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy TNFR mRNAs, the use ofhammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target mRNAhave the following sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence of TNFR-6α (FIG. 1, SEQ ID NO: 1) and TNFR-6β(FIGS. 2A-B, SEQ ID NO:3). Preferably, the ribozyme is engineered sothat the cleavage recognition site is located near the 5′ end of theTNFR mRNA; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express TNFR invivo. DNA constructs encoding the ribozyme may be introduced into thecell in the same manner as described above for the introduction ofantisense encoding DNA. A preferred method of delivery involves using aDNA construct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol III or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous TNFR messages and inhibit translation.Since ribozymes unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

Endogenous gene expression can also be reduced by inactivating or“knocking out” the TNFR gene and/or its promoter using targetedhomologous recombination. (E.g., see Smithies et al., Nature 317:230-234(1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart. The contents of each of the documents recited in this paragraph isherein incorporated by reference in its entirety.

Antibodies according to the present invention may be prepared by any ofa variety of standard methods using TNFR immunogens of the presentinvention. Such TNFR immunogens include the TNFR protein shown in FIGS.1 and 2A-B (SEQ ID NO:2 and SEQ ID NO:4, respectively) (which may or maynot include a leader sequence) and polypeptide fragments of TNFRcomprising the ligand binding and/or extracellular domains of TNFR.

Polyclonal and monoclonal antibody agonists or antagonists according tothe present invention can be raised according to the methods disclosedherein and and/or known in the art, such as, for example, those methodsdescribed in Tartaglia and Goeddel, J. Biol. Chem.267(7):43044307(1992); Tartaglia et al., Cell 73:213-216 (1993), andInternational application publication number WO 94/09137 (the contentsof each of these three applications are herein incorporated by referencein their entireties), and are preferably specific to polypeptides of theinvention having the amino acid sequence of SEQ ID NO:2 and/or SEQ IDNO:4. Antibodies according to the present invention may be prepared byany of a variety of methods described herein, and known in the art.

Further antagonist according to the present invention include solubleforms of TNFR, e.g., TNFR fragments that include the ligand bindingdomain from the extracellular region of the full length receptor. Suchsoluble forms of the receptor, which may be naturally occurring orsynthetic, antagonize TNFR mediated signaling by competing with the cellsurface TNFR for binding to TNF-family ligands and/or antagonize TNFRmediated inhibition of apoptosis by, for example, disrupting the abilityof TNFR to multimerize and/or to bind to and thereby neutralizeapoptosis inducing ligands, such as, for example, Fas ligand and AIM-II.Thus, soluble forms of the receptor that include the ligand bindingdomain are novel cytokines capable of reducing TNFR-mediated inhibitionof tumor necrosis induced by TNF-family ligands. Other such cytokinesare known in the art and include Fas B (a soluble form of the mouse Fasreceptor) that acts physiologically to limit apoptosis induced by Fasligand (Hughes, D. P. and Crispe, I. N., J. Exp. Med. 182:1395-1401(1995)).

Proteins and other compounds which bind the extracellular domains arealso candidate agonist and antagonist according to the presentinvention. Such binding compounds can be “captured” using the yeasttwo-hybrid system (Fields and Song, Nature 340:245-246 (1989)). Amodified version of the yeast two-hybrid system has been described byRoger Brent and his colleagues (Gyuris, J. et al., Cell 75:791-803(1993); Zervos, A. S. et al., Cell 72:223-232 (1993)).

By a “TNF-family ligand” is intended naturally occurring, recombinant,and synthetic ligands that are capable of binding to a member of the TNFreceptor family and inducing the ligand/receptor signaling pathway.Members of the TNF ligand family include, but are not limited to, theTNFR-6α & -6β ligands, TNF-α, lymphotoxin-α (LT-α, also known as TNF-β),LT-β, FasL, CD40, CD27, CD30, 4-1BB, OX40, TRAIL, AIM-II, and nervegrowth factor (NGF).

Formulation and Administration

The TNFR polypeptide composition will be formulated and dosed in afashion consistent with good medical practice, taking into account theclinical condition of the individual patient (especially the sideeffects of treatment with TNFR-6α or -6β polypeptide alone), the site ofdelivery of the TNFR polypeptide composition, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” of TNFR polypeptide forpurposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofTNFR polypeptide administered parenterally per dose will be in the rangeof about 1 μg/kg/day to 10 mg/kg/day of patient body weight, although,as noted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the TNFR polypeptide is typically administered at a doserate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 14injections per day or by continuous subcutaneous infusions, for example,using a mini-pump. An intravenous bag solution may also be employed. Thelength of treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

Effective dosages of the compositions of the present invention to beadministered may be determined through procedures well known to those inthe art which address such parameters as biological half-life,bioavailability, and toxicity. Such determination is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

Bioexposure of an organism to TNFR-6α or -6β polypeptide during therapymay also play an important role in determining a therapeutically and/orpharmacologically effective dosing regime. Variations of dosing such asrepeated administrations of a relatively low dose of TNFR-6α or -6βpolypeptide for a relatively long period of time may have an effectwhich is therapeutically and/or pharmacologically distinguishable fromthat achieved with repeated administrations of a relatively high dose ofTNFR-6α or -6β polypeptide for a relatively short period of time.

Using the equivalent surface area dosage conversion factors supplied byFreireich, E. J., et al. (Cancer Chemotherapy Reports 50(4):219-44(1966)), one of ordinary skill in the art is able to convenientlyconvert data obtained from the use of TNFR-6α or -6β polypeptide in agiven experimental system into an accurate estimation of apharmaceutically effective amount of TNFR-6α or -6β polypeptide to beadministered per dose in another experimental system. Experimental dataobtained through the administration of TNFR6-Fc in mice (see, forinstance, Example 21) may converted through the conversion factorssupplied by Freireich, et al., to accurate estimates of pharmaceuticallyeffective doses of TNFR-6 in rat, monkey, dog, and human. The followingconversion table (Table IV) is a summary of the data provided byFreireich, et al. Table IV gives approximate factors for convertingdoses expressed in terms of mg/kg from one species to an equivalentsurface area dose expressed as mg/kg in another species tabulated.

TABLE IV Equivalent Surface Area Dosage Conversion Factors. TO Mouse RatMonkey Dog Human FROM (20 g) (150 g) (3.5 kg) (8 kg) (60 kg) Mouse 1 ½ ¼⅙   1/12 Rat 2 1 ½ ¼ 1/7 Monkey 4 2 1 ⅗ ⅓ Dog 6 4 5/3 1 ½ Human 12 7 3 21

Thus, for example, using the conversion factors provided in Table IV, adose of 50 mg/kg in the mouse converts to an appropriate dose of 12.5mg/kg in the monkey because (50 mg/kg)×(¼)=12.45 mg/kg. As an additionalexample, doses of 0.02, 0.08, 0.8, 2, and 8 mg/kg in the mouse equate toeffect doses of 1.667 micrograms/kg, 6.67 micrograms/kg, 66.7micrograms/kg, 166.7 micrograms/kg, and 0.667 mg/kg, respectively, inthe human.

TNFR-6 alpha and/or TNFR-6 beta polypeptides of the invention may beadministered using any method known in the art, including, but notlimited to, direct needle injection at the delivery site, intravenousinjection, topical administration, catheter infusion, biolisticinjectors, particle accelerators, gelfoam sponge depots, othercommercially available depot materials, osmotic pumps, oral orsuppositorial solid pharmaceutical formulations, decanting or topicalapplications during surgery, aerosol delivery. Such methods are known inthe art. TNFR-6 alpha and/or TNFR-6 polypeptides of the invention may beadministered as part of a pharmaceutical composition, described in moredetail below. Methods of delivering TNFR-6 alpha and/or TNFR-6 betapolynucleotides of the invention are known in the art and described inmore detail herein.

Pharmaceutical compositions containing the TNFR of the invention may beadministered orally, rectally, parenterally, intracistemally,intravaginally, intraperitoneally, topically (as by powders, ointments,drops or transdermal patch), bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The term “parenteral” as used herein refersto modes of administration which include intravenous, intramuscular,intraperitoneal, intrastemal, subcutaneous and intraarticular injectionand infusion.

The TNFR polypeptide is also suitably administered by sustained-releasesystems. Suitable examples of sustained-release compositions includesuitable polymeric materials (such as, for example, semi-permeablepolymer matrices in the form of shaped articles, e.g., films, ormirocapsules), suitable hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for exanple, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556(1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J.Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech.12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

In a preferred embodiment, compositions of the invention are formulatedin a biodegradable, polymeric drug delivery system, for example asdescribed in U.S. Pat. Nos. 4,938,763; 5,278,201; 5,278,202; 5,324,519;5,340,849; and 5,487,897 and in International Publication NumbersWO01/35929, WO00/24374, and WO00/06117 which are hereby incorporated byreference in their entirety. In specific preferred embodiments thecompositions of the invention are formulated using the ATRIGEL®Biodegradable System of Atrix Laboratories, Inc. (Fort Collins, Colo.).

Examples of biodegradable polymers which can be used in the formulationof compositions of the invention include, but are not limited to,polylactides, polyglycolides, polycaprolactones, polyanhydrides,polyamides, polyurethanes, polyesteramides, polyorthoesters,polydioxanones, polyacetals, polyketals, polycarbonates,polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates,polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates,poly(malic acid), poly(amino acids), poly(methyl vinyl ether),poly(maleic anhydride), polyvinylpyrrolidone, polyethylene glycol,polyhydroxycellulose, chitin, chitosan, and copolymers, terpolymers, orcombinations or mixtures of the above materials. The preferred polymersare those that have a lower degree of crystallization and are morehydrophobic. These polymers and copolymers are more soluble in thebiocompatible solvents than the highly crystalline polymers such aspolyglycolide and chitin which also have a high degree ofhydrogen-bonding. Preferred materials with the desired solubilityparameters are the polylactides, polycaprolactones, and copolymers ofthese with glycolide in which there are more amorphous regions toenhance solubility. In specific preferred embodiments, the biodegradablepolymers which can be used in the formulation of compositions of theinvention are poly(lactide-co-glycolides). Polymer properties such asmolecular weight, hydrophobicity, and lactide/glycolide ratio may bemodified to obtain the desired drug release profile (See, e.g.,Ravivarapu et al., Journal of Pharmaceutical Sciences 89:732-741 (2000),which is hereby incorporated by reference in its entirety).

It is also preferred that the solvent for the biodegradable polymer benon-toxic, water miscible, and otherwise biocompatible. Examples of suchsolvents include, but are not limited to, N-methyl-2-pyrrolidone,2-pyrrolidone, C2 to C6 alkanols, C1 to C15 alchohols, dils, triols, andtetraols such as ethanol, glycerine propylene glycol, butanol; C3 to C15alkyl ketones such as acetone, diethyl ketone and methyl ethyl ketone;C3 to C15 esters such as methyl acetate, ethyl acetate, ethyl lactate;alkyl ketones such as methyl ethyl ketone, C1 to C15 amides such asdimethylformamide, dimethylacetamide and caprolactam; C3 to C20 etherssuch as tetrahydrofuran, or solketal; tweens, triacetin, propylenecarbonate, decylmethylsulfoxide, dimethyl sulfoxide, oleic acid,1-dodecylazacycloheptan-2-one, Other preferred solvents are benzylalchohol, benzyl benzoate, dipropylene glycol, tributyrin, ethyl oleate,glycerin, glycofural, isopropyl myristate, isopropyl palmitate, oleicacid, polyethylene glycol, propylene carbonate, and triethyl citrate.The most preferred solvents are N-methyl-2-pyrrolidone, 2-pyrrolidone,dimethyl sulfoxide, triacetin, and propylene carbonate because of thesolvating ability and their compatibility.

Additionally, formulations comprising compositions of the invention anda biodegradable polymer may also include release-rate modificationagents and/or pore-forming agents. Examples of release-rate modificationagents include, but are not limited to, fatty acids, triglycerides,other like hydrophobic compounds, organic solvents, plasticizingcompounds and hydrophilic compounds. Suitable release rate modificationagents include, for example, esters of mono-, di-, and tricarboxylicacids, such as 2-ethoxyethyl acetate, methyl acetate, ethyl acetate,diethyl phthalate, dimethyl phthalate, dibutyl phthalate, dimethyladipate, dimethyl succinate, dimethyl oxalate, dimethyl citrate,triethyl citrate, acetyl tributyl citrate, acetyl triethyl citrate,glycerol triacetate, di(n-butyl) sebecate, and the like; polyhydroxyalcohols, such as propylene glycol, polyethylene glycol, glycerin,sorbitol, and the like; fatty acids; triesters of glycerol, such astriglycerides, epoxidized soybean oil, and other epoxidized vegetableoils; sterols, such as cholesterol; alcohols, such as C.sub.6-C.sub.12alkanols, 2-ethoxyethanol, and the like. The release rate modificationagent may be used singly or in combination with other such agents.Suitable combinations of release rate modification agents include, butare not limited to, glycerin/propylene glycol, sorbitol/glycerine,ethylene oxide/propylene oxide, butylene glycol/adipic acid, and thelike. Preferred release rate modification agents include, but are notlimited to, dimethyl citrate, triethyl citrate, ethyl heptanoate,glycerin, and hexanediol. Suitable pore-forming agents that may be usedin the polymer composition include, but are not limited to, sugars suchas sucrose and dextrose, salts such as sodium chloride and sodiumcarbonate, polymers such as hydroxylpropylcellulose,carboxymethylcellulose, polyethylene glycol, and polyvinylpyrrolidone.Solid crystals that will provide a defined pore size, such as salt orsugar, are preferred.

In specific preferred embodiments the compositions of the invention areformulated using the BEMA™ BioErodible Mucoadhesive System, MCA™MucoCutaneous Absorption System, SMP™ Solvent MicroParticle System, orBCP™ BioCompatible Polymer System of Atrix Laboratories, Inc. (FortCollins, Colo.). In other specific embodiments, compositions of theinvention are formulated using the ProLease® sustained release sytemavailable from Alkermes, Inc. (Cambridge, Mass.).

Sustained-release compositions also include liposomally entrappedcompositions of the invention (see generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,N.Y., pp. 317 -327 and 353-365 (1989)). Liposomes containing TNFRpolypeptides my be prepared by methods known per se: DE 3,218,121;Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwanget al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal TNFR polypeptide therapy.

In yet an additional embodiment, the compositions of the invention aredelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the TNFR polypeptideis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the TNFRpolypeptide uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The TNFR polypeptide is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of TNFR polypeptide salts.

TNFR polypeptides to be used for therapeutic administration must besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic TNFRpolypeptide compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

TNFR polypeptides ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous TNFR polypeptide solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized TNFR polypeptide using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention may be employed in conjunctionwith other therapeutic compounds.

The compositions of the invention may be administered alone or incombination with other therapeutic agents, including but not limited to,chemotherapeutic agents, anti-opportunistic infection agents,antivirals, antibiotics, steroidal and non-steroidalanti-inflammatories, immunosuppressants, conventional immunotherapeuticagents and cytokines. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the compositions of the invention are administered incombination with other members of the TNF family. TNF, TNF-related orTNF-like molecules that may be administered with the compositions of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, CD27L, CD30L, CD40L, 4-1BBL,DcR3, OX40L, TNF-gamma International application publication number WO96/14328), AIM-I (International application publication number WO97/33899), AIM-II (International application publication number WO97/34911), APRIL (J. Exp. Med. 188(6): 1185-1190), endokine-alpha(International Publication No. WO 98/07880), OPG, and neutrokine-alpha(International application publication number WO 98/18921), TWEAK, OX40,and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27,CD40 and 4-IBB, TR2 (International application publication number WO96/34095), DR3 (International Publication No. WO 97/33904), DR4(International application publication number WO 98/32856), TR5(International application publication number WO 98/30693), TR7(International application publication number WO 98/41629), TRANK, TR9(International application publication number WO 98/56892), TR10(International application publication number WO 98/54202),312C2(International application publication number WO 98/06842), and TR12.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In specific embodiments, compositions of the invention are administeredin combination with immunosuppressants. Immunosuppressants preparationsthat may be administered with the compositions of the invention include,but are not limited to, ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™(cyclosporin), PROGRAF™ (tacrolimus), CELLCEPT™ (mycophenolate),Azathioprine, glucorticosteroids, and RAPAMUNE™ (sirolimus). In aspecific embodiment, immunosuppressants may be used to prevent rejectionof organ or bone marrow transplantation.

In certain embodiments, compositions of the invention are administeredin combination with antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the compositionsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith compositions of the invention to treat AIDS and/or to prevent ortreat HIV infection.

In other embodiments, compositions of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe compositions of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVOIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKNE™(sargramostim/GM-CSF). In a specific embodiment, compositions of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, compositions of the invention are used in any combinationwith ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, compositions of theinvention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™,and/or AZITHROMYCIN™ to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, compositions of the invention are used in any combinationwith GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, compositions of the invention are used in anycombination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, compositions of the invention are used inany combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, compositions of theinvention are used in any combination with PYRIMETHAMINE™ and/orLEUCOVORIN™ to prophylactically treat or prevent an opportunisticToxoplasma gondii infection. In another specific embodiment,compositions of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

In a further embodiment, the compositions of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the compositions of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine

In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, amoxicillin, aminoglycosides, beta-lactam(glycopeptide), beta-lactamases, Clindamycin, chloramphenicol,cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim,trimethoprim-sulfamthoxazole, and vancomycin.

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the compositionsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compostions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the compositions of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, compositions of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In anotherembodiment, compositions of the invention are administered incombination with Rituximab. In a further embodiment, compositions of theinvention are administered with Rituxmab and CHOP, or Rituxmab and anycombination of the components of CHOP.

In an additional embodiment, the compositions of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12,IL13, IL15, anti-CD40, CD40L, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha,and TNF-beta. In another embodiment, compositions of the invention maybe administered with any interleukin, including, but not limited to,IL-1alpha, IL-1beta, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, and IL-21. In a preferred embodiment, the compositions of theinvention are administered in combination with TNF-alpha. In anotherpreferred embodiment, the compositions of the invention are administeredin combination with IFN-alpha.

In an additional embodiment, the compositions of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the compositions of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682 110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PIGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PIGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B-186 (VEGF-B186), as disclosed in InternationalPublication Number WO 96/26736; Vascular Endothelial Growth Factor-D(VEGF-D), as disclosed in International Publication Number WO 98/02543;Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/07832; and Vascular EndothelialGrowth Factor-E (VEGF-E), as disclosed in German Patent NumberDE19639601. The above mentioned references are incorporated herein byreference herein.

In an additional embodiment, the compositions of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the compositions of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In additional embodiments, the compositions of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Chromosome Assays

The nucleic acid molecules of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNAs hereindisclosed are used to clone genomic DNA of a TNFR protein gene. This canbe accomplished using a variety of well known techniques and libraries,which generally are available commercially. The genomic DNA then is usedfor in situ chromosome mapping using well known techniques for thispurpose.

In addition, in some cases, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Fluorescence in situ hybridization (“FISH”) of a cDNA cloneto a metaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance In Man, available on-line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Expression and Purification of TNFR-6 alpha andTNFR-6 beta in E. coli

The bacterial expression vector pQE60 is used for bacterial expressionin this example (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,91311). pQE60 encodes ampicillin antibiotic resistance (“Ampr”) andcontains a bacterial origin of replication (“ori”), an IPTG induciblepromoter, a ribosome binding site (“RBS”), six codons encoding histidineresidues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni—NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that a DNA fragment encoding apolypeptide may be inserted in such as way as to produce thatpolypeptide with the six His residues (i.e., a “6×His tag”) covalentlylinked to the carboxyl terminus of that polypeptide. However, in thisexample, the polypeptide coding sequence is inserted such thattranslation of the six His codons is prevented and, therefore, thepolypeptide is produced with no 6×His tag.

The DNA sequences encoding the desired portions of TNFR-6 alpha andTNFR-6 beta proteins comprising the mature forms of the TNFR-6 alpha andTNFR-6 beta amino acid sequences are amplified from the deposited cDNAclones using PCR oligonucleotide primers which anneal to the aminoterminal sequences of the desired portions of the TNFR-6α or -6βproteins and to sequences in the deposited constructs 3′ to the cDNAcoding sequence. Additional nucleotides containing restriction sites tofacilitate cloning in the pQE60 vector are added to the 5′ and 3′sequences, respectively.

For cloning the mature form of the TNFR-6α protein, the 5′ primer hasthe sequence 5′ CGCCCATGGCAGAAACACCCACCTAC 3′ (SEQ ID NO:19) containingthe underlined NcoI restriction site. One of ordinary skill in the artwould appreciate, of course, that the point in the protein codingsequence where the 5′ primer begins may be varied to amplify a desiredportion of the complete protein shorter or longer than the mature form.The 3′ primer has the sequence 5′ CGCAAGCTTCTCTTTCAGTGCAAGTG 3′ (SEQ IDNO:20) containing the underlined HindIII restriction site. For cloningthe mature form of the TNFR-6β protein, the 5′ primer has the sequenceof SEQ ID NO:19 above, and the 3′ primer has the sequence 5′CGCAAGCTTCTCCTCAGCTCCTGCAGTG 3′ (SEQ ID NO:21) containing the underlinedHindIII restriction site.

The amplified TNFR-6 alpha and TNFR-6 beta DNA fragments and the vectorpQE60 are digested with NcoI and HindIII and the digested DNAs are thenligated together. Insertion of the TNFR-6 alpha and TNFR-6 beta DNA intothe restricted pQE60 vector places the TNFR-6 alpha and TNFR-6 betaprotein coding region including its associated stop codon downstreamfrom the IPTG-inducible promoter and in-frame with an initiating AUG.The associated stop codon prevents translation of the six histidinecodons downstream of the insertion point.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance (“Kanr”),is used in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressingTNFR-6α or -6β protein, is available commercially from QIAGEN, Inc.,supra. Transformants are identified by their ability to grow on LBplates in the presence of ampicillin and kanamycin. Plasmid DNA isisolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6.isopropyl-13-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

To purify the TNFR-6 alpha and TNFR-6 beta polypeptide, the cells arethen stirred for 3-4 hours at 4° C. in 6M guanidine-HCl, pH 8. The celldebris is removed by centrifugation, and the supernatant containing theTNFR-6 alpha and TNFR-6 beta is dialyzed against 50 mM Na-acetate bufferpH 6, supplemented with 200 mM NaCl. Alternatively, the protein can besuccessfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol,25 mM Tris/HCl pH 7.4, containing protease inhibitors. Afterrenaturation the protein can be purified by ion exchange, hydrophobicinteraction and size exclusion chromatography. Alternatively, anaffinity chromatography step such as an antibody column can be used toobtain pure TNFR-6 alpha and TNFR-6 beta protein. The purified proteinis stored at 4° C. or frozen at −80° C.

The following alternative method may be used to purify TNFR-6α or -6βexpressed in E. coli when it is present in the form of inclusion bodies.Unless otherwise specified, all of the following steps are conducted at4-10° C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10° C. and the cells are harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells ware then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GnHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the TNFR-6α or-6β polypeptide-containing supernatant is incubated at 4° C. overnightto allow further GnHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GnHCl solubilized protein is refolded by quickly mixingthe GnHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4° C. without mixing for 12 hours prior tofurther purification steps.

To clarify the refolded TNF receptor polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 μm membranefilter with appropriate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 mm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

Fractions containing the TNF receptor polypeptide are then pooled andmixed with 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the TNFR-6α or -6β polypeptide (determined, for instance, by16% SDS-PAGE) are then pooled.

The resultant TNF receptor polypeptide exhibits greater than 95% purityafter the above refolding and purification steps. No major contaminantbands are observed from Commassie blue stained 16% SDS-PAGE gel when 5μg of purified protein is loaded. The purified protein is also testedfor endotoxin/LPS contamination, and typically the LPS content is lessthan 0.1 ng/ml according to LAL assays.

Example 2 Cloning and Expression of TNFR-6 alpha and TNFR-6 betaProteins in a Baculovirus Expression System

In this illustrative example, the plasmid shuttle vector pA2 is used toinsert the cloned DNA encoding complete protein, including its naturallyassociated secretory signal (leader) sequence, into a baculovirus toexpress the mature TNFR-6α or -6β protein, using standard methods asdescribed in Summers et al., A Manual of Methods for Baculovirus Vectorsand Insect Cell Culture Procedures, Texas Agricultural ExperimentalStation Bulletin No. 1555 (1987). This expression vector contains thestrong polyhedrin promoter of the Autographa californica nuclearpolyhedrosis virus (AcMNPV) followed by convenient restriction sitessuch as BamHI, Xba I and Asp718. The polyadenylation site of the simianvirus 40 (“SV40”) is used for efficient polyadenylation. For easyselection of recombinant virus, the plasmid contains thebeta-galactosidase gene from E. coli under control of a weak Drosophilapromoter in the same orientation, followed by the polyadenylation signalof the polyhedrin gene. The inserted genes are flanked on both sides byviral sequences for cell-mediated homologous recombination withwild-type viral DNA to generate a viable virus that express the clonedpolynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (1989).

The cDNA sequence encoding the full length TNFR-6α or -6β protein in adeposited clone, including the AUG initiation codon and the naturallyassociated leader sequence shown in SEQ ID NO:2 or 4 is amplified usingPCR oligonucleotide primers corresponding to the 5′ and 3′ sequences ofthe gene. The 5′ primer for TNFR-6 alpha and TNFR-6 beta has thesequence 5′ CGCGGATCCGCCATCATGAGGGCGTGGAGGGGCCAG 3′ (SEQ ID NO:22)containing the underlined BamHI restriction enzyme site. All of thepreviously described primers encode an efficient signal for initiationof translation in eukaryotic cells, as described by Kozak, M., J. Mol.Biol. 196:947-950 (1987). The 3′ primer for TNFR-6α has the sequence 5′CGCGGTACCCTCTTTCAGTGCAAGTG 3′ (SEQ ID NO:23) containing the underlinedAsp718 restriction site. The 3′ primer for TNFR-6β has the sequence 5′CGCGGTACCCTCCTCAGCTCCTGCAGTG 3′ (SEQ ID NO:24) containing the underlinedAsp718 restriction site.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with the appropriate restrictionenzyme for each of the primers used, as specified above, and again ispurified on a 1% agarose gel.

The plasmid is digested with the same restriction enzymes andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.).

The fragment and dephosphorylated plasmid are ligated together with T4DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1Blue (Statagene Cloning Systems, La Jolla, Calif.) cells are transformedwith the ligation mixture and spread on culture plates. Bacteria areidentified that contain the plasmid with the human TNF receptor gene bydigesting DNA from individual colonies using the enzymes usedimmediately above and then analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing. This plasmid is designated herein pA2-TNFR-6α or pA2TNFR-6β(collectively pA2-TNFR).

Five μg of the plasmid pA2-TNFR is co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One μg of BaculoGold™ virus DNA and 5 μg of theplasmid pA2-TNFR are mixed in a sterile well of a microtiter platecontaining 50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27° C. The transfection solution is then removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. Cultivation is then continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10). After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C.

To verify the expression of the TNF receptor gene Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus at a multiplicity of infection(“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours laterthe medium is removed and is replaced with SF900 II medium minusmethionine and cysteine (available from Life Technologies Inc.,Rockville, Md.). After 42 hours, 5 μCi of ³⁵S-methionine and 5 μCi³⁵S-cysteine (available from Amersham) are added. The cells are furtherincubated for 16 hours and then are harvested by centrifugation. Theproteins in the supernatant as well as the intracellular proteins areanalyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe mature form of the TNF receptor protein.

Example 3 Cloning and Expression of TNFR-6 alpha and TNFR-6 beta inMammalian Cells

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, hygromycin allows theidentification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulto develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of proteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology,438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart etal., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3′intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

Example 3(a) Cloning and Expression in COS Cells

The expression plasmid, pTNFR-α-HA and -6β-HA, is made by cloning aportion of the cDNA encoding the mature form of the TNF receptor proteininto the expression vector pcDNAI/Amp or pcDNAIII (which can be obtainedfrom Invitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37: 767 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

A DNA fragment encoding the complete TNF receptor polypeptide is clonedinto the polylinker region of the vector so that recombinant proteinexpression is directed by the CMV promoter. The plasmid constructionstrategy is as follows. The TNF receptor cDNA of a deposited clone isamplified using primers that contain convenient restriction sites, muchas described above for construction of vectors for expression of a TNFreceptor in E. coli. Suitable primers can easily be designed by those ofordinary skill in the art.

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith XbaI and EcoRI and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037), and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisor other means for the presence of the fragment encoding the TNFR-α and-6β polypeptides.

For expression of recombinant TNFR-α and -6β, COS cells are transfectedwith an expression vector, as described above, using DEAE-DEXTRAN, asdescribed, for instance, in Sambrook et al., Molecular Cloning: aLaboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor,N.Y. (1989). Cells are incubated under conditions for expression of TNFRby the vector.

Expression of the pTNFR-α-HA and -6β-HA fusion protein is detected byradiolabeling and immunoprecipitation, using methods described in, forexample Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP40, 0.1% SDS, 1%NP40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et alcitedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 3(b) Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of TNFR-6 alpha and TNFR-6beta polypeptides. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr(ATCC Accession No. 37146). The plasmid contains the mouse DHFR geneunder control of the SV40 early promoter. Chinese hamster ovary- orother cells lacking dihydrofolate activity that are transfected withthese plasmids can be selected by growing the cells in a selectivemedium (alpha minus MEM, Life Technologies) supplemented with thechemotherapeutic agent methotrexate. The amplification of the DHFR genesin cells resistant to methotrexate (MTX) has been well documented (see,e.g., Alt, F. W., Kellems, R. M., Bertino, J. R., and Schimke, R. T.,1978, J. Biol. Chem. 253:1357-1370, Hamlin, J. L. and Ma, C. 1990,Biochem. et Biophys. Acta, 1097:107-143, Page, M. J. and Sydenham, M. A.1991, Biotechnology 9:64-68). Cells grown in increasing concentrationsof MTX develop resistance to the drug by overproducing the targetenzyme, DHFR, as a result of amplification of the DHFR gene. If a secondgene is linked to the DHFR gene, it is usually co-amplified andover-expressed. It is known in the art that this approach may be used todevelop cell lines carrying more than 1,000 copies of the amplifiedgene(s). Subsequently, when the methotrexate is withdrawn, cell linesare obtained which contain the amplified gene integrated into one ormore chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985:438-447)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530 (1985)).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, Xba I,and Asp718. Behind these cloning sites the plasmid contains the 3′intron and polyadenylation site of the rat preproinsulin gene. Otherhigh efficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the TNF receptor polypeptide in aregulated way in mammalian cells (Gossen, M., & Bujard, H., Proc. Natl.Acad. Sci. USA 89:5547-5551 (1992)). For the polyadenylation of the mRNAother signals, e.g., from the human growth hormone or globin genes canbe used as well. Stable cell lines carrying a gene of interestintegrated into the chromosomes can also be selected uponco-transfection with a selectable marker such as gpt, G418, orhygromycin. It is advantageous to use more than one selectable marker inthe beginning, e.g., G418 plus methotrexate.

The plasmid pC4 is digested with the restriction enzymes appropriate forthe specific primers used to amplify the TNF receptor of choice asoutlined below and then dephosphorylated using calf intestinalphosphates by procedures known in the art. The vector is then isolatedfrom a 1% agarose gel.

The DNA sequence encoding the TNF receptor polypeptide is amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′sequences of the desired portion of the gene. The 5′ primer for TNFR-6alpha and TNFR-6 beta containing the underlined BamHI site, has thefollowing sequence: 5′ CGCGGATCCGCCATCATGAGGGCGTGGAGGGGCCAG 3′ (SEQ IDNO:22). The 3′ primer for TNFR-6′ has the sequence 5′CGCGGTACCCTCTTTCAGTGCAAGTG 3′ (SEQ ID NO:23) containing the underlinedAsp718 restriction site. The 3′ primer for TNFR-6β has the sequence 5′CGCGGTACCCTCCTCAGCTCCTGCAGTG 3′ (SEQ ID NO:24) containing the underlinedAsp718 restriction site.

The amplified fragment is digested with the endonucleases which will cutat the engineered restriction site(s) and then purified again on a 1%agarose gel. The isolated fragment and the dephosphorylated vector arethen ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells arethen transformed and bacteria are identified that contain the fragmentinserted into plasmid pC4 using, for instance, restriction enzymeanalysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 4 Tissue Distribution of TNF Receptor mRNA Expression

Northern blot analysis is carried out to examine TNFR-6α or -6β geneexpression in human tissues, using methods described by, among others,Sambrook et al., cited above. A cDNA probe containing the entirenucleotide sequence of a TNF receptor protein (SEQ ID NO:1 or 3) islabeled with ³²P using the rediprime™ DNA labeling system (Amersham LifeScience), according to manufacturer's instructions. After labeling, theprobe is purified using a CHROMA SPIN-100™ column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labeled probe is then used to examine varioushuman tissues for TNF receptor mRNA.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) are obtained from Clontech andare examined with the labeled probe using ExpressHyb™ hybridizationsolution (Clontech) according to manufacturer's protocol numberPT1190-1. Following hybridization and washing, the blots are mounted andexposed to film at −70° C. overnight, and films developed according tostandard procedures.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

Example 5 Gene Therapy Using Endogenous TNFR-6 Gene

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous TNFR (i.e., TNFR-6)sequence with a promoter via homologous recombination as described, forexample, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; Internationalapplication publication number WO 96/29411, published Sep. 26, 1996;International application publication number WO 94/12650, published Aug4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989);and Zijistra et al., Nature 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired. Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous TNFR-6, flanking the promoter. The targeting sequence willbe sufficiently near the 5′ end of TNFR-6 so the promoter will beoperably linked to the endogenous sequence upon homologousrecombination. The promoter and the targeting sequences can be amplifiedusing PCR. Preferably,the amplified promoter contains distinctrestriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ endof the first targeting sequence contains the same restriction enzymesite as the 5′ end of the amplified promoter and the 5′ end of thesecond targeting sequence contains the same restriction site as the 3′end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenousTNFR-6 sequence. This results in the expression of TNFR-6 in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the TNFR-6 locus, plasmid pUC18(MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMVpromoter is amplified by PCR with an XbaI site on the 5′ end and a BamHIsite on the 3′ end. Two TNFR-6 non-coding sequences are amplified viaPCR: one TNFR-6 non-coding sequence (TNFR-6 fragment 1) is amplifiedwith a HindIII site at the 5′ end and an Xba site at the 3′ end; theother TNFR-6 non-coding sequence (TNFR-6 fragment 2) is amplified with aBamHI site at the 5′ end and a HindIII site at the 3′ end. The CMVpromoter and TNFR-6 fragments are digested with the appropriate enzymes(CMV promoter—XbaI and BamHI; TNFR-6 fragment 1—XbaI; TNFR-6 fragment2—BamHI) and ligated together. The resulting ligation product isdigested with HindIII, and ligated with the HindII-digested pUC18plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5×10⁶ is thenadded to the cuvette, and the cell suspension and DNA solutions aregently mixed. Electroporation is performed with a Gene-Pulser apparatus(Bio-Rad). Capacitance and voltage are set at 960 μF and 250-300 V,respectively. As voltage increases, cell survival decreases, but thepercentage of surviving cells that stably incorporate the introduced DNAinto their genome increases dramatically. Given these parameters, apulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37° C. The following day, the media is aspiratedand replaced with 10 ml of fresh media and incubated for a further 16-24hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 6 Effect of TNFR in Treating Graft-Versus-Host Disease in Mice

The invention also encompasses a method for the treatment ofrefractory/severe acute GVHD in patients comprising administering to thepatients (preferably human), TNFR polypeptides or TNFR agonists of theinvention.

An analysis of the use of soluble TNFR polypeptides of the invention(e.g., TNFR-6) to treat graft-versus-host disease (GVHD) is performedthrough the use of a C57BL/6 parent into (BALB/c×C57BL/6) F1 mousemodel. This parent into F1 mouse model is a well-characterized andreproducible animal model of GVHD in bone marrow transplant patients,which is well known to one of ordinary skill in the art (see, e.g.,Gleichemann et al, Immunol Today 5:324, 1984, which is hereinincorporated by reference in its entirety). Soluble TNFR is expected tobind to FasL and inhibit FasL-mediated apoptosis, which plays a criticalpathogenic role in the hepatic, cutaneous and lymphoid organ damageobserved in this animal model of GVHD (Baker et al, J. Exp. Med.183:2645, (1996); Charles et al, J. Immunol. 157:5387, (1996); andHattori et al, Blood 91:4051, (1998), each of which is hereinincorporated by reference in its entirety).

Initiation of the GVHD condition is induced by the intravenous injectionof ˜1-3×10⁸ spleen cells from C57BL/6 mice into (BALB/c×C57BL/6) F1 mice(both are available from Jackson Lab, Bar Harbor, Me.). Groups of 6 to 8mice receive either 0.1 to 5.0 mg/kg of TNFR or human IgG isotypecontrol intraperitoneally or intradermally on every other day followingthe injection of spleen cells. The effect of TNFR on liver enzymerelease in the sera, an indicator of liver damage, is analyzed twice perweek for at least 3 weeks. When there is a significant amount of liverenzymes being detected in human IgG-treated mice, the animals aresacrificed for histological evaluation of the relative degree of tissuedamage in the liver, spleen, skin and intestine, and for the therapeuticeffect TNFR has elicited on these organs.

The ability of TNFR to ameliorate systems associated withrefractory/severe acute GVHD is indicated by a reduction of liver enzymerelease in the sera, tissue damage and/or reduced cachexia, loss of bodyweight and/or lethality when compared to the control.

Finally, TNFR- and human IgG-treated animals undergo a clinicalevaluation every other day to assess cachexia, body weight andlethality.

TNFR in combination therapy with TNF-α inhibitors may also be examed inthis GVHD murine model.

Example 7 TNFR-6α (DcR3) Suppresses AIM-II-Mediated Apoptosis

Background

The members of the tumor necrosis factor (TNF) family are involved inregulating diverse biological activities such as regulation of cellproliferation, differentiation, cell survival, cell death, cytokineproduction, lymphocyte co-stimulation, immunoglobulin secretion, andisotype switching (Armitage, R., Curr. Opin. Immunol. 6, 407-413 (1994);Tewari, M. et al., Curr. Opin. Genet. Dev. 6, 39-44 (1996)). Receptorsin this family share a common structural motif in their extracellulardomains consisting of multiple cysteine-rich repeats of approximately 30to 40 amino acids (Gruss, H.-J., et al., Blood 85, 3378-3404 (1995)).While TNFR1, CD95/Fas/APO-1, DR3/TRAMP/APO-3, DR4/TRAIL-R1/APO-2,DR5/TRAIL-R2, and DR6 receptors contain a conserved intracellular motifof 30-40 amino acids called death domain, associated with the activationof apoptotic signaling pathways, other members which contain a lowsequence identity in the intracellular domains, stimulate thetranscription factors NF-κB and AP-1 (Armitage, R., Curr. Opin. Immunol.6, 407-413 (1994); Tewari, M. et al., Curr. Opin. Genet. Dev. 6, 39-44(1996); Gruss, H.-J. et al., Blood 85, 3378-3404 (1995)).

Most TNF receptors contain functional cytoplasmic domain and theyinclude TNFR1 (Loetscher, H et al., Cell 61, 351-356 (1990); Schall, T.J., et al., Cell 61, 361-370 (1990)), TNFR2 (Smith, C. A., et al.,Science 248, 1019-1023 (1990)), lymphotoxin β receptor (LTβR) (Baens,M., et al., Genomics 16, 214-218 (1993)), 4-1BB (Kwon, B. S., et al.,Proc. Natl. Acad. Sci. USA 86, 1963-1967 (1989)), HVEM/TR2/ATAR (Kwon,B. S., et al., J. Biol. Chem. 272, 14272-14276 (1997); Montgomery, R.I., et al., Cell 87, 427-436 (1996); Hsu, H., et al., J. Biol. Chem.272, 13471-13474 (1997)), NGFR (Johnson, D., et al., Cell 47, 545-554(1986)), CD27 (Van Lier, R. A., et al., J. Immunol. 139, 1589-1596(1987)), CD30 (Durkorp, H., et al., Cell 68, 421-427 (1992)), CD40(Banchereau, J., et al., Cell 68, 421-427 (1994)), OX40 (Mallett, S., etal., EMBO J. 9, 1063-1068 (1990)), Fas (Itoh, N., et al., Cell 66,233-243 (1991)), DR3/TRAMP (Chinnaiyan, A. M., et al., Science 274,990-992 (1996)), DR4/TRAIL-R₁ (Pan, G., et al., Science 276, 111-113(1996)), DR5/TRAIL-R2 (Pan, G., et al., Science 277, 815-818) (1997),and RANK (Anderson, D. et al., Nature 390, 175-179 (1997)). Some membersof the TNFR superfamily do not have cytoplasmic domains and aresecreted, such as osteoprotegerin (OPG) (Simmonet, et al., Cell 89,309-319 (1997)), or linked to the membrane through a glycophospholipidtail, such as TRID/DcR1/TRAIL-R3 (Degli-Esposti, M. A., et al., J. Exp.Med. 186, 1165-1170 (1997); Sheridan, J. P., et al., Science 277,818-821 (1997)). Viral open reading frames encoding soluble TNFRs havealso been identified, such as SFV-T2 (Smith, C. A., et al., Science 248,1019-1023 (1990)), Va53 (Howad, S. T., et al., Virology 180, 633-647(1991)), G4RG (Hu, F. Q., et al., Virology 204, 343-356 (1994)), andcrmB (Gruss, H.-J, et al., Blood 85, 3378-3404 (1995)).

By searching an expressed sequence tag (EST) database, a new member ofthe TNFR superfamily was identified, named TNFR-6α, and wascharacterized as a soluble cognate ligand for AIM-II and FasL/CD95L.AIM-II and FasL mediate the apoptosis, which is the most commonphysiological form of cell death and occurs during embryonicdevelopment, tissue remodeling, immune regulation and tumor regression.

AIM-II is highly induced in activated T lymphocytes and macrophages.AIM-II was characterized as a cellular ligand for HVEM/TR2 and LTβR(Mauri, D. N., et al., Immunity 8, 21-30 (1998)). HVEM/TR2 is a receptorfor herpes simplex virus type 1 (HSV-1) entry into human T lymphoblasts.Soluble form of HVEM/TR2-Fc and antibodies to HVEM/TR2 were shown toinhibit a mixed lymphocyte reaction, suggesting a role for this receptoror its ligand in T lymphocyte proliferation (Kwon, B. et al., J. Biol.Chem. 272, 14272-14276 (1997); Mauri, D. N., et al., Immunity, 21-30(1998); Harrop, J. A., et al., J. Immunol. 161, 1786-1794 (1998)). Thelevel of LTβR expression is prominent on epithelial cells but is absentin T and B lymphocytes. Signaling via LTβR triggers cell death in someadenocarcinomas (Browning, J. L., et al., J. Exp. Med. 183, 867-878(1996)). AIM-II produced by activated lymphocytes could evoke immunemodulation from hematopoietic cells expressing only HVEM/TR2, and induceapoptosis of tumor cells, which express both LTβR and HVEM/TR2 receptors(Zhai, Y., et al., J. Clin. Invest. 102, 1142-1151 (1998); Harrop, J.A., et al., J. Biol. Chem. 273, 27548-27556 (1998)).

FasL is one of the major effectors of cytotoxic T lymphocytes andnatural killer cells. It is also involved in the establishment ofperipheral tolerance, in the activation-induced cell death oflymphocytes. Moreover, expression of FasL in nonlymphoid and tumor cellscontributes to the maintenance of immune privilege of tissues bypreventing the infiltration of Fas-sensitive lymphocytes (Nagata, S.,Cell 88, 355-365 (1997)). FasL is also processed and shed from thesurface of human cell (Schneider, P., et al., J. Exp. Med. 187,1205-1213 (1998)).

Here, we demonstrate that TNFR-6α, a new member of the TNFR superfamilybinds AIM-II and FasL. Therefore TNFR-6α, may act as an inhibitor inAIM-II-induced tumor cell death by blocking AIM-II interaction with itsreceptors.

Materials and Methods

Identification and Cloning of New Members of the TNFR Superfamily.

An EST cDNA database, obtained from more than 600 different cDNAlibraries, was screened for sequence homology with the cysteine-richmotif of the TNFR superfamily, using the blastn and tblastn algorithms.Three EST clones containing an identical open reading frame whose aminoacid sequence showed significant homology to TNFR-II were identifiedfrom cDNA libraries of human normal prostate and pancreas tumor. Afull-length TNFR-6 alpha cDNA clone encoding an intact N-terminal signalpeptide was obtained from a human normal prostate library.

RT-PCR Analysis.

For RT-PCR analysis, total RNA was isolated using Trizol (GIBCO) fromvarious human cell lines before and after stimulation with PMA/lonomycinor LPS. RNA was converted to cDNA by reverse transcription and amplifiedfor 35 cycles by PCR. Primers used for amplification of the TNFR-6 alphafragment are according to the sequence of TNFR-6 alpha. β-actin was usedas an internal control for RNA integrity. PCR products were run on 2%agarose gel, stained with ethidium bromide and visualized by UVillumination.

Recombinant Protein Production and Purification.

The recombinant TNFR-6 alpha protein was produced with hexa-histidine atthe C-terminus. TNFR-6 alpha-(His) encoding the entire TNFR-6 alphaprotein was amplified by PCR. For correctly oriented cloning, a HindIIIsite on the 5′ end of the forward primer (5′-AGACCCAAGCTTCCTGCTCCAGCAAGGACCATG-3′: SEQ ID NO:25) and a BamHI site on the 5′ end of thereverse primer (5′-AGACGGGATCCTTAGTGGTGGTGGTGGTGGTGCAC

AGGGAGGAAGCGCTC-3′: SEQ ID NO:26) were created. The amplified fragmentwas cut with HindIII/BamHI and cloned into mammalian expression vector,pCEP4 (Invitrogen). The TNFR-6 alpha-(His)/pCEP4 plasmid was stablytransfected into HEK 293 EBNA cells to generate recombinant TNFR-6alpha-(His). Serum free culture media from cells transfected TNFR-6alpha-(His)/pCEP4 were passed through Ni-column (Novagen). The columneluents were fractionated by SDS-PAGE and TNFR-6 alpha-(His) wasdetected by western blot analysis using the anti-poly(His)₆ antibody(Sigma).

Production of HVEM/TR2-Fc, LTβR-Fc and Flag-tagged soluble AIM-II(soluble AIM-II) fusion proteins were previously described (Zhai, Y., etal., J. Clin. Invest. 102, 1142-1151(1998)). Fc fusionprotein-containing supernatants were filtered and trapped onto protein-GSepharose beads. Flag-tagged soluble AIM-II proteins were purified withanti-FLAG® mAb affinity column.

Immunoprecipitation.

TNFR-6 alpha-(His) was incubated overnight with various Flag-taggedligands of TNF superfamily and anti-FLAG® agarose in binding buffer (150mM NaCl, 0.1% NP40, 0.25% gelatin, 50 mM HEPES, pH 7.4) at 4° C., andthen precipitated. The bound proteins were resolved by 12.5% SDS-PAGEand detected by western blot with HRP-conjugated anti-poly(His)₆ oranti-human IgG1 antibodies.

Cell-Binding Assay.

For cell-binding assays, HEK 293 EBNA cells were stably transfectedusing calcium phosphate method with pCEP4/full sequence of AIM-II cDNAor pCEP4 vector alone. After selection with Hygromycin B, cells wereharvested with 1 mM EDTA in PBS and incubated with TNFR-6 alpha-(His),HVEM/TR2-Fc, or LTβR-Fc for 20 minutes on ice. For detecting Fc-fusionprotein, cells were stained with FITC-conjugated goat anti-human IgG. Todetect TNFR-6 alpha binding, cells were stained with anti-poly(His)₆ andFITC conjugated goat anti-mouse IgG consecutively. The cells wereanalyzed by FACScan (Becton Dickinson).

Cytotoxicity Assay.

Cytotoxicity assays using HT29 cells were carried out as describedpreviously (Browning, J. L., et al., J. Exp. Med. 183, 867-878 (1996)).Briefly, 5000 HT29 cells were seeded in 96-well plates with 1% FBS, DMEMand treated with soluble AIM-II (10 ng/ml) and 10 units/ml humanrecombinant interferon-γ (IFN-γ). Serial dilutions of TNFR-6 alpha-(His)were added in quadruplicate to microtiter wells. Cells treated withIFN-γ and soluble AIM-II were incubated with various amounts of TNFR-6alpha-(His) for 4 days before the addition of [³H]thymidine for the last6 h of culture. Cells were harvested, and thymidine incorporation wasdetermined using a liquid scintillation counter.

Results and Discussion

TNFR-6 Alpha is a New Member of the TNFR Superfamily

TNFR-6 alpha was identified by searching an EST database. Three clonescontaining an identical open reading frame were identified from cDNAlibraries of human normal prostate and pancreas tumor. A full-lengthTNFR-6 alpha cDNA encoding an intact N-terminal signal peptide wasobtained from a human normal prostate library. The open reading frame ofTNFR-6 alpha encodes 300 amino acids. To determine the N-terminal aminoacid sequence of mature TNFR-6 alpha, hexa-histidine tagged TNFR-6 alphawas expressed in mammalian cell expression system and the N-terminalamino acid sequence were determined by peptide sequencing. TheN-terminal sequence of the processed mature TNFR-6 alpha-(His) startedfrom amino acid 30, indicating that the first 29 amino acids constitutedthe signal sequence. Therefore, the mature protein of TNFR-6 alpha wascomposed of 271 amino acids with no transmembrane region. There was onepotential N-linked glycosylation site (Asn173) in TNFR-6 alpha. Like OPG(Simmonet, W. et al., Cell 89, 309-319 (1997)), the predicted proteinwas a soluble, secreted protein and the recombinant TNFR-6 alphaexpressed in mammalian cells was ˜40 kD as estimated on polyacrylamidegel. Alignment of the amino sequences of TNFR-I, TNFR-II, 4-1BB,TR2/HVEM, LTβR, TR1/OPG and TNFR-6 alpha illustrated the existence of apotential cysteine-rich motif. TNFR-6 alpha contained two perfect andtwo imperfect cysteine-rich motifs and its amino acid sequence wasremarkably similar to TR1/OPG amino acid sequence. TNFR-6 alpha shares˜30% sequence homology with OPG and TNFR-II.

mRNA Expression

We analyzed expression of TNFR-6 alpha mRNA in human multiple tissues byNorthern blot hybridization. Northern blot analyses indicated thatTNFR-6 alpha mRNA was ˜1.3 kb in length and was expressed predominantlyin lung tissue and colorectal adenocarcinoma cell line SW480. RT-PCRanalyses were performed to determine the expression patterns of TNFR-6alpha in various cell lines. TNFR-6 alpha transcript was detected weaklyin most hematopoietic cell lines. The expression of TNFR-6 alpha wasinduced upon activation in Jurkat T leukemia cells. Interestingly,TNFR-6 alpha mRNA was constitutively expressed in endothelial cell line,HUVEC at high level.

Identification of the Ligand for TNFR-6 alpha

To identify the ligand for TNFR-6 alpha, several Flag-tagged solubleproteins of TNF ligand family members were screened for binding torecombinant TNFR-6 alpha-(His) protein by immuno-precipitation. TNFR-6alpha-(His) selectively bound AIM-II-Flag and FasL-Flag amongFlag-tagged soluble TNF ligand members tested. This result indicatesthat TNFR-6 alpha binds at least two ligands, AIM-II and FasL. AIM-IIexhibits significant sequence homology with the C-terminalreceptor-binding domain of FasL (31%) but soluble AIM-II is unable tobind to Fas (Mauri, D. N., et al., Immunity 8, 21-30 (1998); Zhai, Y.,et al., J. Clin. Invest. 102, 1142-1151 (1998)). They may have a similarbinding epitope for TNFR-6 alpha binding.

Previously, Zhai and Harrop (Zhai, Y., et al., J. Clin. Invest. 102,1142-1151 (1998); Harrop, J. A., et al., J. Biol. Chem. 273, 27548-27556(1998)) reported the biological functions of AIM-II and its possiblemechanisms of action as a ligand for HVEM/TR2 and/or LTβR. AIM-II isexpressed in activated T cells. AIM-II, in conjunction with serumstarvation or addition of IFN-γ, inhibits the cell proliferation intumor cells, MDA-MB-231 and HT29.

To determine whether TNFR-6 alpha might act as an inhibitor to AIM-IIinteractions with HVEM/TR2 or LTβR, TNFR-6 alpha-(His) was used as acompetitive inhibitor in AIM-II-HVEM/TR2 interaction. When AIM-II wasimmunoprecipitated with HVEM/TR2-Fc in the presence of TNFR-6alpha-(His), HVEM/TR2-Fc binding to AIM-II was decreased competitivelyby TNFR-6 alpha-(His) but TNFR-6 alpha-(His) binding to AIM-II was notchanged by HVEM/TR2-Fc. Furthermore, the binding of HVEM/TR2-Fc (6 nM)or LTβR (6 nM) was completely inhibited by 20 nM of TNFR-6 alpha-(His)protein in immunoprecipitation assays. These results support the notionthat TNFR-6 alpha may act as a strong inhibitor of AIM-II functionthrough HVEM/TR2 and LTβR.

Binding of TNFR-6 alpha-(His) to AIM-II-Transfected Cells

To determine whether TNFR-6 alpha binds to AIM-II expressed on cellsurface, we performed binding assay using AIM-II-transfected HEK 293EBNA cells by flow cytometry. AIM-II-transfected HEK 293 EBNA cells werestained significantly by TNFR-6 alpha-(His) as well as by HVEM/TR2-Fcand LTβR-Fc. No binding was detected by HVEM/TR2-Fc or LTβR-Fc on pCEP4vector-transfected HEK 293 EBNA cells. Furthermore, control isotype didnot bind to AIM-II-transfected HEK 293 EBNA cells, and any of abovefusion proteins did not bind to vector-transfected cells, confirming thespecificity of these bindings. These bindings indicate that TNFR-6 alphacan bind to both soluble and membrane-bound forms of AIM-II.

TNFR-6 alpha Inhibits AIM-II-Induced Cytotoxicity in HT29 Cells

Browning et al. (J. Exp. Med. 183, 867-878 (1996)) have shown that Fasactivation leads to rapid cell death (12-24 h) whereas LTβR takes 2-3days in induction of apoptosis for colorectal adenocarcinoma cell line,HT29. Zhai et al. (J. Clin. Invest. 102, 1142-1151 (1998)) also reportedthat AIM-II leads to the death of the cells expressing both LTβR andHVEM/TR2 but not the cells expressing only the LTβR or HVEM/TR2receptor. Both HVEM/TR2 and LTβR are involved cooperatively inAIM-II-mediated killing of HT29 cells (Zhai, Y., et al., J. Clin.Invest. 102, 1142-1151(1998)).

To determine whether binding of TNFR-6 alpha inhibits AIM-II-mediatedcytotoxicity, HT29 cells were incubated with 10 ng/ml of soluble AIM-IIand IFN-γ (10 U/ml) in the presence of 200 ng/ml of LTβR-Fc or TNFR-6alpha-(His). TNFR-6 alpha-(His) blocked significantly theAIM-II-mediated cell killing. Cells were also incubated with solubleAIM-II and/or IFN-γ in the presence of varying concentration of TNFR-6alpha-(His). TNFR-6 alpha-(His) blocked soluble AIM-II-induced celldeath in a dose-dependent manner. Taken together, TNFR-6 alpha appearsto act as a natural inhibitor of AIM-II-induced tumor cell killing. Thedata also suggest that TNFR-6 alpha contributes to immune evasion oftumors.

AIM-II interaction with HVEM/TR2 and/or LTβR may trigger the distinctbiological events, such as T cell proliferation, blocking ofHVEM-dependent HSV1 infection and anti-tumor activity (Mauri, D. N., etal., Immunity 8, 21-30 (1998); Zhai, Y., et al., J. Clin. Invest. 102,1142-1151 (1998); Harrop, J. A., et al., J. Biol. Chem. 273, 27548-27556(1998)). TNFR-6 alpha may act as an inhibitor of AIM-II interaction andmay play diverse roles in different cell types. TNFR-6 alpha may act asa decoy receptor and contribute to immune evasion both in slow and rapidtumor cell death, that are mediated by AIM-II and/or FasL mediatedapoptosis pathway.

HUVEC cells constitutively expressed TNFR-6 alpha in RT-PCR analysis.AIM-II and FasL have been known to be expressed in activated T cells.Therefore TNFR-6 alpha and its ligands may be important for interactionsbetween activated T lymphocytes and endothelium. TNFR-6 alpha may beinvolved in activated T cell trafficking as well as endothelial cellsurvival.

Example 8 Activation-Induced Apoptosis Assay

Activation-induced apoptosis is assayed using SupT-13 T leukemia cellsand is measured by cell cycle analysis. The assay is performed asfollows. SupT-13 cells are maintained in RPMI containing 10% FCS inlogarithmic growth (about 1×10⁶). Sup-T13 cells are seeded in wells of a24 well plate at 0.5×10⁶/ml, 1 ml/well. AIM II protein or Fas Ligandprotein (0.01, 0.1, 1, 10, 100, 1000 ng/ml) or buffer control is addedto the wells and the cells are incubated at 37° C. for 24 hours in thepresence or absence of the TNFR polypeptides of the invention. The wellsof another 24 well plate are prepared with or without anti-CD3 antibodyby incubating purified BC3 mAb at a concentration of 10 μg/ml insterile-filtered 0.05M bicarbonate buffer, pH 9.5 or buffer alone inwells at 0.5 ml/well. The plate is incubated at 4° C. overnight. Thewells of antibody coated plates are washed 3 times with sterile PBS, at4° C. The treated Sup-T13 cells are transfered to the antibody coatedwells and incubated for 18 hrs., at 37° C. Apoptosis is measured by cellcycle analysis using presidium iodide and flow cytometry. Proliferationof treated cells is measured by taking a total of 300 μl of eachtreatment well and delivering in to triplicate wells (100 μl/well) of 96well plates. To each well add 20 μl/well ³H-thymidine (0.5 μCi/20 μl, 2Ci/mM) and incubate 18 hr., at 37° C. Harvest and count ³H-thymidineuptake by the cells. This measurement may be used to confirm an effecton apoptosis if observed by other methods. The positive controls for theassay is Anti-CD3 crosslinking alone, Fas Ligand alone, and/or AIM-IIalone. In addition, profound and reproducible apoptosis in this lineusing anti-Fas monoclonal antibody (500 ng/ml in soluble form-IgM mAb)has been demonstrated. The negative control for the assay is medium orbuffer alone. Also, crosslinking with another anti-CD3 mAB (OKT3) hasbeen shown to have no effect. TNFR agonists according to the inventionwill demonstrate a reduced apoptosis when compared to the treatment ofthe Sup-T13 cells with AIM-II or Fas Ligand in the absence of the TNFRagonist. TNFR antagonists of the invention can be identified bycombining TNFR polypeptides having Fas Ligand or AIM-II binding affinity(e.g., mature TNFR) with the TNFR polypeptide to be tested andcontacting this combination in solution with AIM-II or Fas Ligand andthe Sup-T13 cells. The negative control for this assay is a mixturecontaining the mature TNFR, Sup-T13 cells, and AIM-II or Fas Ligand(FasL) alone. Samples containing TNFR antagonists of the invention willdemonstrate increased apoptosis when compared to the negative control.

If an effect is observed by cell cycle analysis the cells can be furtherstained for the TUNEL assay for flow cytometry or with Annexin V, usingtechniques well known to those skilled in the art.

Example 9 Blocking of Fas Ligand Mediated Apoptosis of Jurkat T-cells byTNFR6 alpha-Fc

Methods.

Jurkat T-cells which express the Fas receptor were treated either withsFas ligand alone or with sFas ligand in combination with Fas-Fc, orTNFR6 alpha-Fc (corresponding to the full length TNFR 6 alpha protein(amino acids 1-300 of SEQ ID NO:2) fused to an Fc domain, as describedherein). The sFas ligand protein utilized was obtained from AlexisCorporation and contains a FLAG® epitope tag at its N-terminus. As ithas been demonstrated previously that cross-linking of Fas ligandutilizing the monoclonal FLAG® epitope enhances significantly theability of Fas ligand to mediate apoptosis, the anti-FLAG® antibody wasincluded in this study. Specifically, 106 Jurkat cells (RPMI+5% serum)were treated with Fas ligand (Alexis) (20 ng/ml) and anti-FLAG® Mab (200ng/ml) and then incubated at 37° C. for 16 hrs. When TNFR6 alpha-Fc wasincluded in the assay, the receptor was preincubated with the Fas ligandand anti-FLAG® Mab for 15 mins.

Results

After incubation, cells were harvested, resuspended in PBS and subjectedto Flow Cytometric Analyses (Table V). In the absence of Fas ligand(FasL), approximately 1% of cells appear to be undergoing apoptosis asmeasured by high annexin staining and poor propidium iodide staining(Table V). Treatment with soluble Fas ligand alone resulted in anapproximate 7-fold increase in the number of apoptotic cells which asexpected could be blocked in the presence of Fas-Fc. Similar to Fas-Fc,TNFR6 alpha-Fc was also capable of blocking Fas mediated apoptosis withthe blocking by TNFR6 alpha-Fc observed in a dose dependent manner overthree logarithmic scales (Table V). The ability of TNFR6 alpha-Fc toblock Fas mediated killing of Jurkat cells was also determined in a celldeath assay (FIGS. 7A-B). In this assay, cells were again treated withcombinations of Fas ligand and TNFR6 alpha-Fc for 16 hrs. To measure thelevels of viable cells after treatment, cells were incubated for 5 hrswith 10% ALOMAR blue and examined spectrophotometrically at OD 570nm-630 nm. Treatment with Fas ligand resulted in a 50% decrease in cellviability (FIGS. 7A-B). The decrease in cell viability can be overcomeby either Fas-Fc or TNFR6 alpha-Fc but not TR5-Fc (FIGS. 7A-B),confirming the ability of TNFR6 alpha to interfere with Fas ligandmediated activity. The ability of TNFR6 alpha-Fc at both 100 ng/ml andat 10 ng/ml to block Fas ligand mediated activity in this assay isstatistically different (p<0.05) than when no TNFR6 alpha-Fc is added(FIGS. 7A-B). Furthermore, the ability of TNFR6 alpha-Fc to block Fasligand mediated cell death and apoptosis appears to be as efficient withFas-Fc (Table V and FIGS. 7A-B).

Table V. FACS Analysis Revealing Blocking of Fas Ligand MediatedApoptosis:

10⁶ Jurkat cells (RPMI+5% serum) were treated with Fas ligand (Allexis;20 ng/ml) and anti-FLAG® antibody (200 ng/ml) and then incubated at 37°C. for 16 hours. When Fc receptor was included in the assay, thereceptor was preincubated with the Fas ligand and anti-FLAG® antibodyMab for 15 minutes. After incubation, cells were harvested, resuspendedin PBS and subjected to Flow Cytometric Analyses.

Treatment % Cells undergoing apoptosis Control (buffer) 1.24 FasL (20ng) 8.87 FasL (20 ng) + Fas-Fc (100 ng) 1.78 FasL (20 ng) + TNFR6alpha-Fc (100 ng) 1.24 FasL (20 ng) + TNFR6 alpha-Fc (10 ng) 2.79 FasL(20 ng) + TNFR6 alpha-Fc (1 ng) 7.95 FasL (20 ng) + TNFR6 alpha-Fc (0.1ng) 8.58Conclusions.

TNFR6 alpha-Fc appears to block Fas ligand mediated apoptosis of Jurkatcells in a dose dependent manner as effectively as Fas ligand.

Example 10 Protein Fusions of TNFR-6 alpha and/or TNFR-6 beta

TNFR-6 alpha and/or TNFR-6 beta polypeptides of the invention areoptionally fused to other proteins. These fusion proteins can be usedfor a variety of applications. For example, fusion of TNFR-6 alphaand/or TNFR-6 beta polypeptides to His-tag, HA-tag, protein A, IgGdomains, and maltose binding protein facilitates purification. (See EP A394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusionto IgG-1, IgG-3, and albumin increases the halflife time in vivo.Nuclear localization signals fused to TNFR-6 alpha and/or TNFR-6 betapolypeptides can target the protein to a specific subcellularlocalization, while covalent heterodimer or homodimers can increase ordecrease the activity of a fusion protein. Fusion proteins can alsocreate chimeric molecules having more than one function. Finally, fusionproteins can increase solubility and/or stability of the fused proteincompared to the non-fused protein. All of the types of fusion proteinsdescribed above can be made using techniques known in the art or byusing or routinely modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also preferably contain convenient restrictionenzyme sites that will facilitate cloning into an expression vector,preferably a mammalian expression vector.

For example, if the pC4 (Accession No. 209646) expression vector isused, the human Fc portion can be ligated into the BamHI cloning site.Note that the 3′ BamHI site should be destroyed. Next, the vectorcontaining the human Fc portion is re-restricted with BamHI, linearizingthe vector, and TNFR-6 alpha and/or TNFR-6 beta polynucleotide, isolatedby the PCR protocol described in Example 1, is ligated into this BamHIsite. Note that the polynucleotide is cloned without a stop codon,otherwise a fusion protein will not be produced.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., International application publication number WO 96/34891.)

Human IgG Fc Region:

(SEQ ID NO:27)GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 11 Production of an Antibody

Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing TR6-alpha and/or TR6-beta are administered toan animal to induce the production of sera containing polyclonalantibodies. In a preferred method, a preparation of TR6-alpha and/orTR6-beta protein is prepared and purified to render it substantiallyfree of natural contaminants. Such a preparation is then introduced intoan animal in order to produce polyclonal antisera of greater specificactivity.

Monoclonal antibodies specific for protein TR6-alpha and/or TR6-beta areprepared using hybridoma technology. (Kohler et al., Nature 256:495(1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al.,Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: MonoclonalAntibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)).In general, an animal (preferably a mouse) is immunized with TR6-alphaand/or TR6-beta polypeptide or, more preferably, with a secretedTR6-alpha and/or TR6-beta polypeptide-expressing cell. Suchpolypeptide-expressing cells are cultured in any suitable tissue culturemedium, preferably in Earle's modified Eagle's medium supplemented with10% fetal bovine serum (inactivated at about 56° C.), and supplementedwith about 10 g/l of nonessential amino acids, about 1,000 U/ml ofpenicillin, and about 100 μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP2O), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981). The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the TR6-alpha and/orTR6-beta polypeptide.

Alternatively, additional antibodies capable of binding to TR6-alphaand/or TR6-beta polypeptide can be produced in a two-step procedureusing anti-idiotypic antibodies. Such a method makes use of the factthat antibodies are themselves antigens, and therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, protein specific antibodies are used to immunize ananimal, preferably a mouse. The splenocytes of such an animal are thenused to produce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theTR6-alpha and/or TR6-beta protein-specific antibody can be blocked byTR6-alpha and/or TR6-beta. Such antibodies comprise anti-idiotypicantibodies to the TR6-alpha and/or TR6-beta protein-specific antibodyand are used to immunize an animal to induce formation of furtherTR6-alpha and/or TR6-beta protein-specific antibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric and humanized antibodies are known in theart and are discussed infra. (See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature314:268 (1985).)

Isolation of Antibody Fragments Directed Against TR6-alpha and/orTR6-beta from a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againstTR6-alpha and/or TR6-beta to which the donor may or may not have beenexposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein byreference in its entirety).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 109 E. coli harboring thephagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 withshaking. Five ml of this culture is used to innoculate 50 ml of2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 μg/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing.

Example 12 Method of Determining Alterations in the TNFR-6 alpha and/orTNFR-6 beta Gene

RNA is isolated from entire families or individual patients presentingwith a phenotype of interest (such as a disease). cDNA is then generatedfrom these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:1. Suggested PCRconditions consist of 35 cycles at 95° C. for 30 seconds; 60-120 secondsat 52-58° C.; and 60-120 seconds at 70° C., using buffer solutionsdescribed in Sidransky, D., et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons ofTNFR-6 alpha and/or TNFR-6 beta are also determined and genomic PCRproducts analyzed to confirm the results. PCR products harboringsuspected mutations in TNFR-6 alpha and/or TNFR-6 beta is then clonedand sequenced to validate the results of the direct sequencing.

PCR products of TNFR-6 alpha and/or TNFR-6 beta are cloned into T-tailedvectors as described in Holton, T. A. and Graham, M. W., Nucleic AcidsResearch, 19:1156 (1991) and sequenced with T7 polymerase (United StatesBiochemical). Affected individuals are identified by mutations in TNFR-6alpha and/or TNFR-6 beta not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in the TNFR-6 alpha and/or TNFR-6 beta gene. Genomic clonesisolated using techniques known in the art are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson, et al., Methods Cell Biol. 35:73-99(1991). Hybridization with the labeled probe is carried out using a vastexcess of human cot-1 DNA for specific hybridization to the TNFR-6 alphaand/or TNFR-6 beta genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C— and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson, et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region of TNFR-6 alpha and/or TNFR-6 beta(hybridized by the probe) are identified as insertions, deletions, andtranslocations. These TNFR-6 alpha and/or TNFR-6 beta alterations areused as a diagnostic marker for an associated disease.

Example 13 Method of Detecting Abnormal Levels of TNFR-6 alpha and/orTNFR-6 beta in a Biological Sample

TNFR-6 alpha and/or TNFR-6 beta polypeptides can be detected in abiological sample, and if an increased or decreased level of TNFR-6alpha and/or TNFR-6 beta is detected, this polypeptide is a marker for aparticular phenotype. Methods of detection are numerous, and thus, it isunderstood that one skilled in the art can modify the following assay tofit their particular needs.

For example, antibody-sandwich ELISAs are used to detect TNFR-6 alphaand/or TNFR-6 beta in a sample, preferably a biological sample. Wells ofa microtiter plate are coated with specific antibodies to TNFR-6 alphaand/or TNFR-6 beta, at a final concentration of 0.2 to 10 ug/ml. Theantibodies are either monoclonal or polyclonal and are produced usingtechnique known in the art. The wells are blocked so that non-specificbinding of TNFR-6 alpha and/or TNFR-6 beta to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining TNFR-6 alpha and/or TNFR-6 beta. Preferably, serial dilutionsof the sample should be used to validate results. The plates are thenwashed three times with deionized or distilled water to remove unboundedTNFR-6 alpha and/or TNFR-6 beta.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate(NPP) substrate solution is then added to each well and incubated 1 hourat room temperature to allow cleavage of the substrate and flourescence.The flourescence is measured by a microtiter plate reader. A standardcurve is prepared using the experimental results from serial dilutionsof a control sample with the sample concentration plotted on the X-axis(log scale) and fluorescence or absorbance on the Y-axis (linear scale).The TNFR-6 alpha and/or TNFR-6 beta polypeptide concentration in asample is then interpolated using the standard curve based on themeasured flourescence of that sample.

Example 14 Method of Treating Decreased Levels of TNFR-6 alpha and/orTNFR-6 beta

The present invention relates to a method for treating an individual inneed of a decreased level of TNFR-6 alpha and/or TNFR-6 beta biologicalactivity in the body comprising, administering to such an individual acomposition comprising a therapeutically effective amount of TNFR-6alpha and/or TNFR-6 beta antagonist. Preferred antagonists for use inthe present invention are TNFR-6 alpha and/or TNFR-6 beta-specificantibodies.

Moreover, it will be appreciated that conditions caused by a decrease inthe standard or normal expression level of TNFR-6 alpha and/or TNFR-6beta in an individual can be treated by administering TNFR-6 alphaand/or TNFR-6 beta, preferably in a soluble and/or secreted form. Thus,the invention also provides a method of treatment of an individual inneed of an increased level of TNFR-6 alpha and/or TNFR-6 betapolypeptide comprising administering to such an individual apharmaceutical composition comprising an amount of TNFR-6 alpha and/orTNFR-6 beta to increase the biological activity level of TNFR-6 alphaand/or TNFR-6 beta in such an individual.

For example, a patient with decreased levels of TNFR-6 alpha and/orTNFR-6 beta polypeptide receives a daily dose 0.1-100 ug/kg of thepolypeptide for six consecutive days. Preferably, the polypeptide is ina soluble and/or secreted form.

Example 15 Method of Treating Increased Levels of TNFR-6 alpha and/orTNFR-6 beta

The present invention also relates to a method for treating anindividual in need of an increased level of TNFR-6 alpha and/or TNFR-6beta biological activity in the body comprising administering to such anindividual a composition comprising a therapeutically effective amountof TNFR-6 alpha and/or TNFR-6 beta or an agonist thereof.

Antisense technology is used to inhibit production of TNFR-6 alphaand/or TNFR-6 beta. This technology is one example of a method ofdecreasing levels of TNFR-6 alpha and/or TNFR-6 beta polypeptide,preferably a soluble and/or secreted form, due to a variety ofetiologies, such as cancer.

For example, a patient diagnosed with abnormally increased levels ofTNFR-6 alpha and/or TNFR-6 beta is administered intravenously antisensepolynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days.This treatment is repeated after a 7-day rest period if the isdetermined to be well tolerated.

Example 16 Method of Treatment Using Gene Therapy—Ex vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing soluble and/or mature TNFR-6 alpha and/or TNFR-6 betapolypeptides, onto a patient. Generally, fibroblasts are obtained from asubject by skin biopsy. The resulting tissue is placed in tissue-culturemedium and separated into small pieces. Small chunks of the tissue areplaced on a wet surface of a tissue culture flask, approximately tenpieces are placed in each flask. The flask is turned upside down, closedtight and left at room temperature over night. After 24 hours at roomtemperature, the flask is inverted and the chunks of tissue remain fixedto the bottom of the flask and fresh media (e.g., Ham's F12 media, with10% FBS, penicillin and streptomycin) is added. The flasks are thenincubated at 37 degree C. for approximately one week.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding TNFR-6 alpha and/or TNFR-6 beta can be amplified usingPCR primers which correspond to the 5′ and 3′ end encoding sequencesrespectively. Preferably, the 5′primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform E. coli HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector containsproperly inserted TNFR-6 alpha and/or TNFR-6 beta.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the TNFR-6 alpha and/or TNFR-6 beta gene is then added to themedia and the packaging cells transduced with the vector. The packagingcells now produce infectious viral particles containing the TNFR-6 alphaand/or TNFR-6 beta gene (the packaging cells are now referred to asproducer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether TNFR-6 alphaand/or TNFR-6 beta protein is produced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 17 Method of Treatment Using Gene Therapy—in vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) TNFR-6 alpha and/or TNFR-6 beta sequences into ananimal to increase or decrease the expression of the TNFR-6 alpha and/orTNFR-6 beta polypeptide. The TNFR-6 alpha and/or TNFR-6 betapolynucleotide may be operatively linked to a promoter or any othergenetic elements necessary for the expression of the TNFR-6 alpha and/orTNFR-6 beta polypeptide by the target tissue. Such gene therapy anddelivery techniques and methods are known in the art, see, for example,International Application publication number WO90/11092, InternationalApplication publication number WO98/11779; U.S. Pat. Nos. 5,693,622,5,705,151, 5,580,859; Tabata H. et al., Cardiovasc. Res. 35:470-479(1997); Chao J. et al., Pharmacol. Res. 35:517-522 (1997); Wolff J. A.Neuromuscul. Disord. 7:314-318 (1997); Schwartz B. et al., Gene Ther.3:405-411 (1996); Tsurumi Y. et al., Circulation 94:3281-3290 (1996)(incorporated herein by reference).

The TNFR-6 alpha and/or TNFR-6 beta polynucleotide constructs may bedelivered by any method that delivers injectable materials to the cellsof an animal, such as, injection into the interstitial space of tissues(heart, muscle, skin, lung, liver, intestine and the like). The TNFR-6alpha and/or TNFR-6 beta polynucleotide constructs can be delivered in apharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the TNFR-6 alpha and/or TNFR-6 beta polynucleotidesmay also be delivered in liposome formulations (such as those taught inFelgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and AbdallahB. et al. (1995) Biol. Cell 85(1):1-7) which can be prepared by methodswell known to those skilled in the art.

The TNFR-6 alpha and/or TNFR-6 beta polynucleotide vector constructsused in the gene therapy method are preferably constructs that will notintegrate into the host genome nor will they contain sequences thatallow for replication. Any strong promoter known to those skilled in theart can be used for driving the expression of DNA. Unlike other genetherapies techniques, one major advantage of introducing naked nucleicacid sequences into target cells is the transitory nature of thepolynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The TNFR-6 alpha and/or TNFR-6 beta polynucleotide construct can bedelivered to the interstitial space of tissues within the an animal,including of muscle, skin, brain, lung, liver, spleen, bone marrow,thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis, ovary, uterus, rectum, nervoussystem, eye, gland, and connective tissue. Interstitial space of thetissues comprises the intercellular fluid, mucopolysaccharide matrixamong the reticular fibers of organ tissues, elastic fibers in the wallsof vessels or chambers, collagen fibers of fibrous tissues, or that samematrix within connective tissue ensheathing muscle cells or in thelacunae of bone. It is similarly the space occupied by the plasma of thecirculation and the lymph fluid of the lymphatic channels. Delivery tothe interstitial space of muscle tissue is preferred for the reasonsdiscussed below. They may be conveniently delivered by injection intothe tissues comprising these cells. They are preferably delivered to andexpressed in persistent, non-dividing cells which are differentiated,although delivery and expression may be achieved in non-differentiatedor less completely differentiated cells, such as, for example, stemcells of blood or skin fibroblasts. In vivo muscle cells areparticularly competent in their ability to take up and expresspolynucleotides.

For the naked TNFR-6 alpha and/or TNFR-6 beta polynucleotide injection,an effective dosage amount of DNA or RNA will be in the range of fromabout 0.05 g/kg body weight to about 50 mg/kg body weight. Preferablythe dosage will be from about 0.005 mg/kg to about 20 mg/kg and morepreferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as theartisan of ordinary skill will appreciate, this dosage will varyaccording to the tissue site of injection. The appropriate and effectivedosage of nucleic acid sequence can readily be determined by those ofordinary skill in the art and may depend on the condition being treatedand the route of administration. The preferred route of administrationis by the parenteral route of injection into the interstitial space oftissues. However, other parenteral routes may also be used, such as,inhalation of an aerosol formulation particularly for delivery to lungsor bronchial tissues, throat or mucous membranes of the nose. Inaddition, naked TNFR-6 alpha and/or TNFR-6 beta polynucleotideconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The dose response effects of injected TNFR-6 alpha and/or TNFR-6 betapolynucleotide in muscle in vivo is determined as follows. SuitableTNFR-6 alpha and/or TNFR-6 beta template DNA for production of mRNAcoding for TNFR-6 alpha and/or TNFR-6 beta polypeptide is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The TNFR-6 alpha and/or TNFR-6 beta template DNA is injectedin 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle overone minute, approximately 0.5 cm from the distal insertion site of themuscle into the knee and about 0.2 cm deep. A suture is placed over theinjection site for future localization, and the skin is closed withstainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 umcross-section of the individual quadriceps muscles is histochemicallystained for TNFR-6 alpha and/or TNFR-6 beta protein expression. A timecourse for TNFR-6 alpha and/or TNFR-6 beta protein expression may bedone in a similar fashion except that quadriceps from different mice areharvested at different times. Persistence of TNFR-6 alpha and/or TNFR-6beta DNA in muscle following injection may be determined by Southernblot analysis after preparing total cellular DNA and HIRT supernatantsfrom injected and control mice. The results of the above experimentationin mice can be use to extrapolate proper dosages and other treatmentparameters in humans and other animals using TNFR-6 alpha and/or TNFR-6beta naked DNA.

Example 18 Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of TNFR-6 alpha and/or TNFR-6 beta onischemia, a rabbit hindlimb ischemia model is created by surgicalremoval of one femoral arteries as described previously (Takeshita, S.et al., Am J. Pathol 147:1649-1660 (1995)). The excision of the femoralartery results in retrograde propagation of thrombus and occlusion ofthe external iliac artery. Consequently, blood flow to the ischemic limbis dependent upon collateral vessels originating from the internal iliacartery (Takeshita, et al., Am J. Pathol 147:1649-1660 (1995)). Aninterval of 10 days is allowed for post-operative recovery of rabbitsand development of endogenous collateral vessels. At 10 daypost-operatively (day 0), after performing a baseline angiogram, theinternal iliac artery of the ischemic limb is transfected with 500 mgnaked TNFR-6 alpha and/or TNFR-6 beta expression plasmid by arterialgene transfer technology using a hydrogel-coated balloon catheter asdescribed (Riessen, R. et al., Hum Gene Ther. 4:749-758 (1993); Leclerc,G. et al., J. Clin. Invest. 90: 936-944 (1992)). When is used in thetreatment, a single bolus of 500 mg protein or control is delivered intothe internal iliac artery of the ischemic limb over a period of 1 min.through an infusion catheter. On day 30, various parameters are measuredin these rabbits: (a) BP ratio—The blood pressure ratio of systolicpressure of the ischemic limb to that of normal limb; (b) Blood Flow andFlow Reserve—Resting FL: the blood flow during undilated condition andMax FL: the blood flow during fully dilated condition (also an indirectmeasure of the blood vessel amount) and Flow Reserve is reflected by theratio of max FL: resting FL; (c) Angiographic Score—This is measured bythe angiogram of collateral vessels. A score is determined by thepercentage of circles in an overlaying grid that with crossing opacifiedarteries divided by the total number m the rabbit thigh; (d) Capillarydensity—The number of collateral capillaries determined in lightmicroscopic sections taken from hindlimbs.

The studies described in this example test activity in TNFR-6 proteins.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides (e.g., gene therapy),agonists, and/or antagonists of TNFR-6 alpha and/or TNFR-6 beta.

Example 19 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

A Diabetic db+/db+ Mouse Model.

To demonstrate that TNFR-6 accelerates the healing process, thegenetically diabetic mouse model of wound healing is used. The fullthickness wound healing model in the db+/db+ mouse is a wellcharacterized, clinically relevant and reproducible model of impairedwound healing. Healing of the diabetic wound is dependent on formationof granulation tissue and re-epithelialization rather than contraction(Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G.et al., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979);Coleman, D. L., Diabetes 31 (Suppl): 1-6 (1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J. Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andwere 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

TNFR-6 alpha and/or TNFR-6 beta is administered using at a rangedifferent doses of TNFR-6 protein, from 4 mg to 500 mg per wound per dayfor 8 days in vehicle. Vehicle control groups received 50 mL of vehiclesolution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) TNFR-6 alpha and/or TNFR-6beta.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 was 64 mm², the corresponding size of the dermalpunch. Calculations were made using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with TNFR-6. This assessment included verification of thepresence of cell accumulation, inflammatory cells, capillaries,fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometeris used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer served as a positive tissue controland human brain tissue is used as a negative tissue control. Eachspecimen included a section with omission of the primary antibody andsubstitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

B. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, S. M. Glucocorticoids andWound healing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahl, S. M. et al., J. Immunol. 115: 476-481(1975); Werb, Z. et al., J. Exp. Med. 147:1684-1694 (1978)).Glucocorticoids retard wound healing by inhibiting angiogenesis,decreasing vascular permeability (Ebert, R. H., et al., An. Intern. Med.37:701-705 (1952)), fibroblast proliferation, and collagen synthesis(Beck, L. S. et al., Growth Factors. 5: 295-304 (1991); Haynes, B. F. etal., J. Clin. Invest. 61: 703-797 (1978)) and producing a transientreduction of circulating monocytes (Haynes, B. F., et al., J. Clin.Invest. 61: 703-797 (1978); Wahl, S. M., “Glucocorticoids and woundhealing”, In: Antiinflammatory Steroid Action: Basic and ClinicalAspects, Academic Press, New York, pp. 280-302 (1989)). The systemicadministration of steroids to impaired wound healing is a well establishphenomenon in rats (Beck, L. S. et al., Growth Factors. 5: 295-304(1991); Haynes, B. F., et al., J. Clin. Invest. 61: 703-797 (1978);Wahl, S. M., “Glucocorticoids and wound healing”, In: AntiinflammatorySteroid Action: Basic and Clinical Aspects, Academic Press, New York,pp. 280-302 (1989); Pierce, G. F. et al., Proc. Natl. Acad. Sci. USA 86:2229-2233 (1989)).

To demonstrate that TNFR-6 alpha and/or TNFR-6 beta can accelerate thehealing process, the effects of multiple topical applications of TNFR-6on full thickness excisional skin wounds in rats in which healing hasbeen impaired by the systemic administration of methylprednisolone isassessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and were 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On theday of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1-5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson caliper. Woundsare considered healed if granulation tissue was no longer visible andthe wound is covered by a continuous epithelium.

TNFR-6 alpha and/or TNFR-6 beta is administered using at a rangedifferent doses of TNFR-6 protein, from 4 mg to 500 mg per wound per dayfor 8 days in vehicle. Vehicle control groups received 50 mL of vehiclesolution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Four groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) were evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) TNFR-6 treated groups.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1was 64 mm², the corresponding size of the dermal punch. Calculationswere made using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffm embedded blocksare sectioned perpendicular to the wound surface (5 mm) and cut using anOlympus microtome. Routine hematoxylin-eosin (H&E) staining wasperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin was improved by treatmentwith TNFR-6 alpha and/or TNFR-6 beta. A calibrated lens micrometer isused by a blinded observer to determine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

The studies described in this example test activity in TNFR-6 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides (e.g., gene therapy),agonists, and/or antagonists of TNFR-6 alpha and/or TNFR-6 beta.

Example 20 Lymphadema Animal Model

The purpose of this experimental approach is to create an appropriateand consistent lymphedema model for testing the therapeutic effects ofin lymphangiogenesis and re-establishment of the lymphatic circulatorysystem in the rat hind limb. Effectiveness is measured by swellingvolume of the affected limb, quantification of the amount of lymphaticvasculature, total blood plasma protein, and histopathology. Acutelymphedema is observed for 7-10 days. Perhaps more importantly, thechronic progress of the edema is followed for up to 3-4 weeks.

Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately ˜350 g aredosed with Pentobarbital. Subsequently, the right legs are shaved fromknee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH.Blood is drawn for serum total protein testing. Circumference andvolumetric measurements are made prior to injecting dye into paws aftermarking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsalpaw). The intradermal dorsum of both right and left paws are injectedwith 0.05 ml of 1% Evan's Blue. Circumference and volumetricmeasurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is madecircumferentially allowing the femoral vessels to be located. Forcepsand hemostats are used to dissect and separate the skin flaps. Afterlocating the femoral vessels, the lymphatic vessel that runs along sideand underneath the vessel(s) is located. The main lymphatic vessels inthis area are then electrically coagulated or suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosisand adductors) are bluntly dissected. The popliteal lymph node is thenlocated.

The 2 proximal and 2 distal lymphatic vessels and distal blood supply ofthe popliteal node are then and ligated by suturing. The popliteal lymphnode, and any accompanying adipose tissue, is then removed by cuttingconnective tissues.

Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (AJ Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of 0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw are measured before operation and daily for 7 days. The effectplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perameter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limbmovement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople and the readings are averaged. Readings are taken from bothcontrol and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetizedwith Pentobarbital and are tested prior to surgery. For dailyvolumetrics animals are under brief halothane anesthetic (rapidimmobilization and quick recovery), both legs are shaved and equallymarked using waterproof marker on legs. Legs are first dipped in water,then dipped into instrument to each marked level then measured by Buxcoedema software(Chen/Victor). Data is recorded by one person, while theother is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serumseparated prior to surgery and the conclusion to the experiment tomeasure for total protein and Ca2+ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared fortissue collection. The limbs were amputated using a quillitine, thenboth experimental and control legs were cut at the ligature and weighed.A second weighing is done as the tibio-cacaneal joint is disarticulatedand the foot is weighed.

Histological Preparations: The transverse muscle located behind the knee(popliteal) area is dissected and arranged in a metal mold, filled withfreezeGel, dipped into cold methylbutane, placed into labeled samplebags at −80 degree C. until sectioning. Upon sectioning, the muscle wasobserved under fluorescent microscopy for lymphatics. Otherimmuno/histological methods are currently being evaluated.

The studies described in this example test activity in TNFR-6 proteins.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides (e.g., gene therapy),agonists, and/or antagonists of TNFR-6 alpha and/or TNFR-6 beta.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

Example 21 TNFR6-Fc Inhibits FasL Mediated Toxicity in a ConA MouseModel of Liver Injury

The intravenous administration of Concanavalin A to mice activates Tlymphocytes and induces both apoptotic and necrotic cell death ofhepatocytes, mimicking aspects of the pathophysiology of chronic activehepatitis (Tiegs et al., J. Clin. Invest. 90: 196. (1992)). Fas-Fcprotein, a dimeric form of Fas expected to inhibit Fas ligand activity,has been reported to reduce liver injury in this model via inhibition ofFas ligand demonstrating an involvement of Fas pathway in the pathology(Ksontini et al., J Immunol.; 60(8):4082-4089 (1998)).

Validation of Model:

To validate the ConA mouse model, Con A was administered intravenouslyto Balb/c mice at 10, 15, and 20 mg/kg dose of ConA along with placeboor Fas-Fc at 97.5 micrograms/mouse. 10 Balb/c mice were used pertreatment group. The mice were sacrificed 22 hours after treatment,serum collected and biochemical analysis performed using a ClinicalChemistry Analyzer ILAB900 (Instrumentation Laboratory) to determine thelevels of the liver specific transaminases, alanine aminotransferase(ALT) and aspartate aminotransferase (AST), which are released in theserum upon liver damage ((Tiegs et al., J. Clin. Invest. 90: 196.(1992)). The administration of FasFc at a dose of 97.5 micrograms/mouse(about 5 mg/kg) was found to significantly inhibit the elevated liverenzymes at ConA doses of 10 and 15 mg/kg but not at 20 mg/kg (data notshown), thus validating the model.

Balb/C mice were injected intravenously with ConA (15 mg/kg) togetherwith or without a three log dose of TNFR6-Fc (0.6, 6. 60 ug/mouse). TheTNFR6-Fc fusion protein used in this example corresponds to the fulllength TNFR-6 alpha polypeptide sequence (amino acids 1-300 of SEQIDNO:2) fused to an Fc domain. 10 Balb/c mice were used per treatmentgroup. The mice were sacrificed 22 hours after treatment and serumlevels of ALT and AST were determined using a Clinical ChemistryAnalyzer ILAB900 (Instrumentation Laboratory). The administration ofTNFR6-Fc significantly inhibited both ALT and AST levels at the highestdose tested (60 micrograms/mouse, 3 mg/kg) by 50% (data not shown). ThusTNFR6-Fc significantly reduced ConA induced serum AST and ALT in a doseresponse fashion.

Effect of TNFR6-Fc on ConA Induced Apoptotic Events in the Liver

Since the elevation in serum liver enzyme levels reflects both apoptoticand non-apoptotic pathways of hepatocyte destruction, a more criticaldetermination of the extent of liver injury can be derived via directmeasurement of apoptotic events. Thus apoptosis was analyzed using wholeliver cell suspensions isolated from mice treated with TNFR6-Fc and ConA. Three independent markers of apoptosis were assessed on the samesample. These include changes in surface expression ofphosphatidylserine, measurements of DNA damage, and caspase activation.

Balb/C mice were injected intravenously with ConA (15 mg/kg) togetherwith or without a three log dose of TNFR6-Fc (0.6, 6. 60 ug/mouse). Cellsuspensions were isolated from the livers of 3 mice/group and livercells were isolated by placing the intact liver tissue on a 70 μm cellstrainer and teased apart with the stopper of a 5 cc syringe using RPMI1640/10% FBS. To remove red blood cells and large piece of tissuedebris, the filtered cell suspension was layered over lymphocyteseparation medium (density 1.0770 g/ml). The interface layer wascollected, washed and the cells were counted. Prior to FACS analysis,the cell suspension was refiltered over a 40 μm filter.

For measurement of Annexin V binding (an indicator of apoptosis), cellswere first incubated with fluorochrome-conjugated monoclonal antibodiesCD45 CyChrome and B220 or anti-TCRβ PE (Pharmingen, San Diego, Calif.).Cells were washed with binding buffer (Pharmingen) then incubated withAnnexin V FITC (Pharmingen). Stained cells were acquired and analyzedusing a Becton Dickinson FACScan (Becton Dickinson, San Jose, Calif.).Only CD45 positive events were collected. Cells staining brightly forB220 and Annexin V were considered apoptotic B cells; cells stainingbrightly for anti-TCRβ and Annexin V were considered apoptotic T cells.

The level of DNA degradation (another hallmark of apoptosis) wasdetermined by Terminal UTP nick-end labeling (TUNEL) which measures thisdegradation by using TdT enzyme to add FITC-labeled dUTP to the 3′ endsof nicked DNA using the Apo-DIRECT kit (Pharmingen) according tomanufacturer's directions. Briefly, cells were fixed in 1%paraformaldhyde, washed in PBS and then fixed with ice-cold 70% ethanol.Cells were washed twice in washing buffer, then incubated with stainingsolution containing TdT enzyme and dUTP-FITC at 37° C. for one hour.Cells were washed twice with rinsing buffer, re-suspended in propidiumiodide solution and acquired on the FACScan. For analysis, an electronicgate was set on singlet events, and cells staining brightly fordUTP-FITC were considered apoptotic cells.

To determine the presence of the active form of caspase-3 (an earlyindicator of apoptosis) cells were incubated in IC FIX (BioSourceInternational, Camarillo, Calif.), washed twice in PBS, thenpermeablized with IC PERM (BioSource). Cells were incubated with 5 μgrabbit anti-caspase-3 PE (Pharmingen) in IC PERM, washed in IC PERM,then washed twice with PBS. Cells were acquired on the FACScan andanalyzed for PE mean fluorescence.

For all three indicators of apoptosis, TNFR6-Fc inhibited apoptosis inlivers of mice as compared to mice treated with Con A alone (Table VI).Using DNA damage as a marker and TUNEL analysis, a dose-dependent trendof inhibition with TR6-Fc was observed. These data support a role forTNFR6-Fc in inhibition of apoptosis in ConA-induced hepatitis.

TABLE VI Apoptosis of liver cells isolated from TNFR6-Fc-treated mice.¹Percent % Apoptotic Cells measured by: Annexin Annexin Treatment TunelCaspase-3 V/TcRβ V/B220 Untreated Control — 18.7 2.0 6.1 Con A (15mg/kg) Control 24.4 35.2 7.0 12.2  TNFR6-Fc (0.6 μg/mouse) 15.6 22.7 3.56.3 TNFR6-Fc (6.0 μg/mouse) 13.6 22.5 2.3 2.9 TNFR6-Fc (60 μg/mouse) 9.5 20.3 3.0 4.2 ¹Liver cell suspensions were analyzed for apoptosisusing one of three independent measures. DNA degradation was measuredusing TUNEL staining; caspase activation by the analysis of the activeform of caspase-3; and annexin V staining of surface membrane changes.Cell suspensions were isolated from the livers of 3 mice/group andpooled. The resulting pooled suspension was used to perform eachanalysis. For Annexin-V staining, only liver CD45+ cells were acquiredand Annexin-V staining assessed on cells costained for B220 or TcRβ.Conclusion:

The findings that TNFR6-Fc reduced both ConA induced serum AST and ALTlevels and ConA induced liver cell apoptosis supports the therapeuticapplication of TNFR-6 alpha and TNFR-6 beta polypeptides of theinvention for the treatment and/or prevention of hepatitis and otherforms of liver injury.

Example 22 In vitro and in vivo Inhibition of FasL Mediated Killing byTNFR-6 alpha

Fas (CD95/Apo1) and Fas ligand (FasL/CD95L), are a pair of pro-apoptoticmediators of the TNF receptor and ligand family that induce apoptosisupon receptor/ligand engagement. Fas/FasL-mediated apoptosis is a normaland important homeostatic mechanism useful in the down-regulation ofhyper-immune responses and the deletion of activated lymphocytes.Fas/FasL-induced apoptosis is also important in host protection andsurveillance, preventing damage to immune privileged sites, andeliminating virus-infected or transformed cells. While necessary fornormal physiological processes, unregulated apoptosis mediated by theFas/FasL system is implicated in organ-specific tissue injury both inexperimental animal models and several human disease states.

This example describes the synthesis and biological activity of a TNFR-6alpha (in this Example, hereinafter “TR6”) fusion protein produced usingthe full length coding region of TR6 and an Fc domain of IgG1.Biochemical and biological characterization of this TR6-Fc form revealedit to, not only bind FasL and inhibit apoptosis in-vitro, but also toblock the mortality associated with iv injection of cross-linked FasLinto Fas⁺ mice. This is the first demonstration of TR6-mediatedinhibition of FasL activity in an in-vivo model. These results show thetherapeutic potential of TR6-Fc in diseases where Fas/FasL is implicatedin mediating organ damage.

Methods of Example 22

Animals

Female Balb/c mice (20-25 g) were obtained from Charles RiverLaboratories (Raleigh, N.C.). Female MLR/lpr mice (30-35 g) wereobtained from Jackson Laboratories (Bar Harbor, Me.). Mice were housedfive per cage, and kept under standard conditions for one week beforebeing used in experiments. The animals were maintained according toNational Research Council standards for the care and use of laboratoryanimals. The animal protocols used in this study were reviewed andapproved by the HGS Institutional Animal Care and Use Committee.

Human TR6-Fc, TR6-Non Fc and Fas-Fc Expression Vectors

Cells infected with baculovirus clone, pA2Fc:TR6 (M1-H300), were grownin media containing 1% ultra low IgG serum. Conditioned culturesupernatant (20 L) was adjusted to pH 7.0, filtered through 0.22 micronfilter and loaded on a Protein A column (BioSepra Ceramic HyperD)previously conditioned with 20 mM phosphate buffer with 0.5 M NaCl,pH7.2. The column was washed with 15 CV of 20 mM phosphate buffercontaining 0.5 M NaCl, pH 7.2, and followed by 5 CV of 0.1 M citric acid(pH 5.0). TR6-Fc eluted with 0.1 M citric acid (pH 2.4)/20% glycerol,and fractions were neutralized with 1M Tris-HCl, pH 9.2. The humanTR6-Fc positive fractions were determined by SDS-PAGE. The peakfractions were pooled and concentrated using an Amicon concentrator. TheTR6-Fc concentrate was then loaded onto a Superdex 200 column containingPBS containing 0.5 M NaCl (Pharmacia) and TR6-Fc positive fractionsdetermined by non-reducing SDS-PAGE. The TR6-Fc positive fractionseluting as disulfide-linked dimers were pooled and further concentratedwith CentriPlus 10K cutoff spin concentrators.

The TR6-Fc protein bound to the Protein A resin contained bothdisulfide-linked Fc dimers and higher disulfide-linked aggregates.Aggregates were removed by Superdex 200 size-exclusion chromatography.The typical yield for TR6-Fc was ˜2 mg/L culture supernatant having apurity of 98% by Reverse-Phase HPLC assay and 92% by N-terminal sequenceassay. The N-terminus started at residue Val 30. The pure proteinbehaved as disulfide linked dimer and was biologically active as itbound FasL in a BIAcore assay to a degree comparable to Fas-Fc.

To confirm purity, TR6-Fc protein was blotted to a ProBlott membranecartridge (PE Biosystems, Inc). After staining with Ponceau S (0.2% in4% acetic acid), the membrane was placed in a “Blot Cartridge”, andsubjected to N-terminal amino acid sequence analysis using a modelABI-494 sequencer (PE Biosystems, Inc.) and the Gas-phase Blot cycles.Proteins were subject to reverse-phase HPLC (Beckmann) analysis toaccess purity. In the case of Fas-Fc the N-terminus was deblocked usingpyroglutamate aminopeptidase ( ) followed by N-terminal sequenceanalysis.

Human Fas(M1-G169)-Fc fusion protein was purified from CHO conditionedmedia by capture on a Poros 50 Protein A affinity column with elution at0.1M citrate pH 2.0 as described for TR6-Fc. Further puriifcation waseffected by size separation on a Superdex-200 gel filtration resin inPBS/glycerol. N-terminal sequence of Fas-Fc was blocked and proteinidentity was confirmed post digestion with pyroglutamate aminopeptidaseto deblock the N-terminus and 16% SDS-PAGE, respectively. The proteinbehaved as disulfide linked dimer as expected for a Fc fusion protein.

BIAcore Chip Preparation and Analysis

The extra-cellular portion of FasL (Oncogene Research Products), aminoacids 103-281, were dialyzed against 10 mM sodium acetate buffer, pH 5and a BIAcore flow cell prepared having 2020 RU of FasL. TR6-Fc andFas-Fc fusion proteins were analyzed at 5 ug/mL in 50 uL HBS buffer andwere injected onto the FasL chip at a flow rate of 15 ul per minute.After injection of the sample the flow cell was equilibrated with HBSand amount of net bound protein determined.

In vitro Soluble Human FasL Mediated Cytotoxicity

The HT-29 cell line, a human colon adenocarcinoma cell line obtainedfrom the ATCC (code ATCC HTB-38) is sensitive to FasL mediatedcytotoxicity, presumably through activation of its Fas receptor. HT-29cells were grown in D-MEM/10% FBS/2 mM Glutamine/pen/strep. To measureFLAG-FasL induced cytotoxicity, target cells were trypsinized, washedand plated in a 96-well plate at 50,000 cells/well. HT-29 cells weretreated with cross-linked FLAG-FasL+anti-FLAG® antibody (1 ng/ml), orwith cross-linked FLAG-FasL in combination with Fas-Fc, or TR6. Althoughuncross-linked FasL can induce cytotoxity in this assay, antibodycross-linking of FasL via its FLAG® domain significantly enhances theability of FasL to mediate apoptosis, and thus the anti-FLAG® antibodywas included. The final volume in each well was 200 ul. After 5 days ofculture, the plate was harvested and 20 ul of Alamar Blue reagent added.To assess final viability, cells were incubated for four hours and theplate analyzed in a CytoFluor fluorescence plate reader with excitationof 530 nm and emission of 590 nm.

The Jurkat human T cell line, which also expresses the Fas receptor, andis sensitive to FasL, was tested in an in vitro cytotoxicity assaysimilar to that used on HT-29 cells. In addition, Jurkat cells wereevaluated by FACS analysis in an apoptosis assay. Jurkat cells (RPMI+5%serum) were seeded at 50,000 cells per well were then treated withFLAG-FasL and anti-FLAG® mouse monoclonal antibody (200 ng/ml) andincubated at 37 C for 16 hrs to induce apoptosis. When TR6 or Fas-Fc wasincluded in the assay, the decoy receptor protein was pre-incubated withFasL and anti-FLAG® antibody for 15 mins. To determine the degree ofapoptosis, cells were harvested, stained with annexin and propidiumiodide and evaluated using FACS analysis.

In vitro Membrane Bound Murine FasL Mediated Cytotoxicity

To analyze the in vitro killing of Fas⁺ target cells by murine FasL,murine effector L929 cells (2.5×10⁵ cells/well) were transfected withmurine FasL and incubated with Fas⁺ murine A20 target cells (5×10³cells/well) labeled with Eu DTPA. After an 18 hour incubation at aneffector:target cell ratio of 50:1, cells were centrifuged, and %release of Eu DTPA quantified as a measure of cell death.

In vivo Cross-Linked FLAG-FasL Induced Mortality

Soluble human FLAG-FasL was synthesized at HGS. To induce cross-linking,FasL was incubated with anti-FLAG® antibody (Sigma, St Louis, Mo.) andinjected iv into mice following a variation of the procedure used bySchneider et al. Fc-fusion proteins were injected iv or sc at varioustime points prior to FasL injection, and mortality recorded over time.Liver samples one centimeter square, were fixed in 10% neutral bufferedformalin for 24 hours, then transferred to 70 percent methanol untiltime for embedding in paraffin. Sections were stained with H&E, andevaluated histologically. Blood was drawn from the heart and used in themeasurement of serum AST and ALT levels.

Statistics

Statistical difference between groups was determined using a Student'sunpaired t test. Error bars represent S.E.M.

Results of Example 22

BIA Core Analysis of TR6-Fc Binding to FasL

BIAcore chip technology provides the opportunity to identify andcharacterize ligands that bind to a given receptor, in this case TR6.The protein ligand can be immobilized and challenged with TR6 tocalculate relative binding units (RU). Conversely, the TR6 receptor canbe immobilized and exposed to various ligands to identify proteins withan affinity for the TR6 receptor.

BIAcore technology was used to determine if human TR6-Fc displayed anybinding to human FasL immobilized on a BIAcore chip. The resultsindicated that TR6-Fc bound to FasL with the same affinity as the Fasreceptor, approximately 100 RU. As a control, TR6-Fc interaction withanother TNF ligand, BLyS, was examined. No significant binding wasfound.

To show the specificity of TR6-Fc for FasL, soluble FLAG-FasL was usedto compete with the immobilized FasL for binding of TR6-Fc. Increasingconcentrations of FasL-Flag were able to inhibit binding of TR6-Fc toimmobilized FasL. At a concentration of 8 ug/ml, FasL-Flag inhibitedbinding of TR6-Fc (2 ug/ml) by 50 percent. When 17 ug/ml of FasL-Flagwas used, inhibition rose to 75 percent.

When TR6-Fc was immobilized, and trimerized FLAG-FasL used as thesoluble protein, the Kd of TR6-Fc was 4.6×10⁻⁹ M, similar to the7.4×10⁻⁹ M Kd for FasFc. TR6 without the Fc portion had a fourfoldreduction in affinity for FasL-Flag with a Kd of 1.7×10⁻⁸ M.

In vitro Effect of TR6 on Soluble Human FasL Mediated Cytotoxicity

The results of this experiment demonstrate the ability of TR6 to blockcross-linked FLAG-FasL mediated HT-29 cell death. FLAG-FasL inducedHT-29 cytotoxity in a dose-dependent manner, with the maximal effect ata concentration between 1 and 10 ng/ml. In the presence of TR6-Fc (1ug/ml), FasL failed to induce cell killing, in agreement with theproposed decoy receptor function of TR6. Unlike TR6-Fc, Fas-Fc did nottotally abrogate FLAG-FasL mediated cell death, but did shift thecytotoxicity curve about 10 fold to the right. TR6-non-Fc also inhibitedFasL mediated killing, but was not as potent as the Fc fusion protein. Anumber of other members of the TNF receptor family, such as TNFR1-Fc,LTBR-Fc, TR2-Fc, TR4-Fc, TR7-Fc, TR8-Fc, TR9-Fc, TR10-Fc and TR11-Fcwere also tested in this assay and failed to block FasL induced killingof HT-29 cells. In a different cytotoxity assay involving the eponymousTNF family member, TR6-Fc failed to inhibit TNFa-induced killing of L929target cells.

The ability of TR6 to block antibody cross-linked FLAG-FasL killing invitro was also observed using human Jurkat cells in a similarcytotoxicity assay. Treatment with FasL at 10 ng/ml resulted in an 80%decrease in cell viability as measured by fluorescence at 530/590.Fas-Fc as well as TR6-Fc and non-Fc significantly reduced FasL-inducedcytotoxicity whether the decoy receptor level was kept constant and FasLincreased, or the FasL level kept constant and the decoy receptorincreased. In both assay systems TR6-Fc appeared to be at least 100 foldmore potent than Fas-Fc.

In another Jurkat cell assay, treatment with FLAG-FasL resulted in anapproximate 7-fold increase in the number of apoptotic cells overuntreated controls, as measured by FACS analysis of annexin staining.FasL-mediated apoptosis was significantly reduced in a dose dependantfashion in the presence of TR6-Fc or Fas-Fc.

In vitro Effect of TR6 on Membrane Bound Murine FasL MediatedCytotoxicity

In an assay using Fas⁺murine A20 target cells labeled with Eu DTPA,TR6-Fc at a concentration of 10 ng/ml, completely inhibited killing bymurine L929 cells transfected with murine FasL. In this assay, the IC₅₀for both TR6-Fc and non-Fc was approximately 1 ng/ml. The potency of TR6in this assay was 100 fold greater than that of Fas-Fc, which had anIC₅₀ of approximately 100 ng/ml. This assay demonstrated that the humanTR6 protein was capable of recognizing, binding to, and blocking, thecytotoxic activity of murine FasL.

The in vivo Effect of TR6-Fc on FLAG-FasL-Induced Mortality in Mice

Female Balb/c mice (n=20) were injected iv with 13 ug of FLAG-FasL mixedwith 50 ug of murine antibody to FLAG. Half of the mice also received aniv injection of 96 ug of TR6-Fc, one hour prior to administration ofcross-linked FLAG-FasL. Since TR6-Fc has a molecular weight of about60,000 compared to 18,500 for FLAG-FasL, this resulted in aTR6-Fc:FLAG-FasL molar ratio of 2.3:1. However, each molecule of TR6-Fcis capable of binding two molecules of FasL.

Within one hour of FasL injection, all the mice injected only withcross-linked FLAG-FasL were dead. The hypotension was so great, that noblood could be obtained for analysis of serum alanine (ALT) andaspartate (AST) aminotransferase levels. In another group of miceinjected with FLAG-FasL uncross-linked by anti-FLAG® antibody, therewere no deaths. There were also no deaths in the cross-linked FLAG-FasLgroup treated with TR6-Fc. Analysis of blood drawn from mice 24 hoursafter TR6-Fc injection, showed an elevated, but not significantly higherserum AST level compared to normal controls (Normal=81±12 units/l;TR6-Fc=548±193 units/l)

To determine its minimum lethal dose, 1, 3, 6 or 13 ug of FLAG-FasL wasmixed with 4, 12, 25 or 50 ug of anti-FLAG® antibody and injected ivinto Balb/c mice (n=3). Only the mice injected with the lowest, 1 ugdose of FLAG-FasL survived. The mean serum AST level 24 hours afterinjection was 2663±1373 compared to the mean normal value of 67±17. Theminimum lethal dose of cross-linked FLAG-FasL appeared to be about 3ug/mouse.

To establish that the in vivo mechanism of FLAG-FasL-induced lethalitywas the binding of FasL to its cell bound Fas receptor, 5 ug ofFLAG-FasL was mixed with 19 ug of anti-FLAG® antibody and injected intoFas⁻ MRL/lpr mice and their Fas⁺ littermates. Within 30 minutes, all theFas⁺ control mice were dead, while all the Fas⁻ MRL/lpr mice survived.This indicated that in vivo FLAG-FasL killing was dependent on theexpression of Fas receptor on the target cells.

In a dose response experiment, TR6-Fc was injected iv at a dose of 2, 8or 24 ug/mouse, one hour before iv injection with FLAG-FasL (4 ug/mouse)mixed with 12 ug of anti-FLAG® antibody (Table VII). This results in aTR6-Fc:FLAG-FasL molar ratio of approximately 1:6, 1:2 and 2:1respectively. In the FLAG-FasL control group, all but one of the tenmice died within two hours. The low dose of TR6-Fc (2 ug) had noprotective activity, all the animals dying within two hours. The middledose of TR6-Fc (8 ug) prolonged life an additional two hours. Only thehigh, 24 ug dose of TR6-Fc was efficacious, with seven of the ten micesurviving without weight loss to the end of the experiment on Day 7.This indicates not only that TR6-Fc was efficacious at a molar ratio of2:1, but also that its protective effect was not lost over time. Incontrast to the activity of TR6-Fc, the non Fc version of TR6 did notreduce FLAG-FasL (4 ug) induced mortality even when injected at 70ug/mouse, a molar ratio of 11:1 (data not shown).

To determine if TR6-Fc exhibited protective activity when injected sc,as opposed to iv, 350 ug of TR6-Fc was injected sc, 1.5, 3 or 5 hoursbefore iv injection of 4 ug of FLAG-FasL mixed with 15 ug of anti-FLAG®antibody (Table VIII). Even at the receptor:ligand molar ratio of 27:1,none of the animals injected sc with TR6-Fc survived for more than twohours, while all of the animals injected iv with 93 ug of TR6-Fc orFas-Fc survived. A different member of the TNF receptor superfamily,TR-11 (93 ug/mouse, iv) was used as a negative control, and failed toprotect any animals from FLAG-FasL induced death. Analysis of blooddrawn from mice, injected iv with TR-6-Fc+FasL showed no significantelevation of AST or ALT levels compared to normal controls.

TABLE VII Dose dependant effect of TR6-Fc (iv) on cross-linked FLAG-FasLinduced mortality Groups (n = 10) Time/% Survival (ug/mouse) <2 Hrs <4Hrs 1 Day 4 Days 7 Days Normal 100 100 100 100 100 FLAG-FasL (3) + anti-10 10 10 10 10 FLAG ® Ab (12) FasL + Ab + TR6- 0 0 0 0 0 Fc (2) FasL +Ab + TR6- 100 10 10 10 10 Fc (8) FasL + Ab + TR6- 90 80 80 70 70 Fc (24)TR6-Fc and/or FLAG-FasL+anti-FLAG® antibody was injected iv into femaleBalb/c mice as described in the Material and Methods.

TABLE VIII Effect of TR6-Fc (sc, iv) and Fas-Fc (iv) on cross-linkedFLAG-FasL induced mortality Time/% Survival Groups <2 Hours >24 HoursNormal 100 100 FLAG-FasL (4 μg/mouse) + 0 0 anti-FLAG ® Ab (15 μg/mouse) TR6Fc (350 μg/mouse) 0 0 Sc, −5 hr TR6Fc (350 μg/mouse) 0 0 sc,−3 hr TR6Fc (350 μg/mouse) 0 0 sc, −1.5 hr TR6Fc (93 μg/mouse) 100 100iv, −1 hr Fas-Fc (93 μg/mouse) 100 100 iv, −1 hr TR11-Fc (93 μg/mouse) 00 iv, −1 hrAll groups except normal controls received an iv injection ofFLAG-FasL+anti-FLAG® antibody at Time 0.

Example 23 Modulation of T Cell Responses By TNFR6: Soluble TNFR6Inhibits Alloactivation and Heart Allograft Rejection

The ability of TNFR6 to interact with LIGHT and the role of TNFR6 inmodulating T cell activities and immunological responses that may beassociated with LIGHT were analyzed according to the experimentsdetailed below.

Materials and Methods of Example 23

Mice

Twelve week-old female C57BL/6 (B6, H-2^(b)), BALB/c, and BALB/c×C57BL/6F1 (H-2^(b×d)) were purchased from Jackson Laboratory (Bar Harbor, Me.)or Charles River (LaSalle, Quebec, Canada). 2C TCR transgenic mice werebred in an animal facility as described in Chen, H., et al., 1996. J.Immunol. 157:4297, which is hereby incorporated by reference in itsentirety.

Expression and Purification of the Human TR6-Fc Fusion Protein

Full-length human TNFR-6 alpha cDNA (FIG. 1, aa 1-300; referred in thisexample hereafter as “TR6”) was PCR-amplified using gene specificprimers, fused to the sequence coding for the Fc domain of human IgG₁and subcloned into a baculovirus expression vector pA2. The constructwas named pA2-Fc:TR6. Sf9 cells infected pA2-Fc:TR6 were grown in media(100 L) containing 1% ultra low IgG serum (100 L). Conditioned culturesupernatant from a bioreactor was harvested by continuous flowcentrifugation. The pH of the supernatant was adjusted to pH 7.0,filtered through 0.22 um filter and loaded on to a Protein A column(BioSepra Ceramic HyperD, Life Technologies, Rockville, Md. 30 ml bedvolume) previously conditioned with 20 mM phosphate buffer, 0.5 M NaCl(pH 7.2). The column was washed with 15 column volumes (CV) of 20 mMphosphate buffer (pH 7.2) containing 0.5 M NaCl followed by 5 CV of 0.1M sodium citrate (pH 5.0). TR6-Fc was eluted with 0.1 M citric acid (pH2.4), and 2 mL fractions were collected into tubes containing 0.6 mlTris-HCl (pH 9.2). The TR6-Fc positive fractions were determined bySDS-PAGE. The peak fractions were pooled and concentrated with a ProteinA column (7 mL bed volume) as described above. The concentrated TR6-Fcwas loaded onto a Superdex 200 column (Amersham Pharmacia, Piscataway,N.J. 90 ml bed volume) and eluted with PBS containing 0.5 M NaCl. TR6-Fcpositive fractions were determined by non-reducing SDS-PAGE. The pooledpositive fractions were dialyzed against 12.5 mM HEPES buffer, pH 5.75containing 50 mM NaCl. The dialysate was then passed through a 0.2 mfilter (Minisart, Sartorius AG, Goettingen, Germany) followed by aQ15X-anion exchange membrane (Sartobind membrane, Sartorius AG,Goettingen, Germany).

Expression and Purification of Full-Length Human TR6 (without Fc)

The full-length TR6 cDNA was PCR-amplified and cloned in to thebaculovirus expression vector pA2 as describe above. Sf9 cells wereinfected with the viral construct, and the culture supernatant of theinfected cells was loaded onto a Poros HS-50 column (Applied Biosystems,Foster City, Calif.) equilibrated in a buffer containing 50 mM Tris-HCl,pH 7, and 0.1M NaCl. The column was washed with 0.1 M NaCl and elutedstepwise with 0.3M, 0.5M, and 1.5M NaCl. The eluded fractions wereanalyzed by SDS-PAGE, and the 0.5 M NaCl fraction containing TR6 proteinwas diluted and loaded onto a set of Poros HQ-50/CM-20 columns in atandem mode. TR6 was eluted from the CM column with a linear gradientfrom 0.2M to 1.0 M NaCl.

Expression and Purification of Human TR2-Fc, MCIF-Fc, and Fas-Fc FusionProteins

The cDNA sequences coding for the extracellular domain of TR2 (aa1-192), the extracellular domain of Fas (aa 1-169) and a beta chemokineMCIF (aa 1-92) were fused with the cDNA sequence coding for the Fcdomain of human IgG₁, and cloned into a eukaryotic expression vectorpC4. The construct was stablely transfected into CHO cells. The Fcfusion proteins from the CHO supernatant were purified with methods usedfor TR6-Fc.

Expression and Purification of the Human LIGHT Protein

The coding sequence of the natural secreted form of LIGHT (aa 83-240)was cloned into a prokaryotic expression vector pHE4 (ATCC DepositNumber 209645, described in U.S. Pat. No. 6,194,168), and expressed inE. coli. Inclusion bodies from the transformed bacteria were dissolvedfor 48-72 hours at 4° C. in 3.5 M guanidine hydrochloride containing 100mM Tris-HCl, pH 7.4 and 2 mM CaCl₂. The solution was quickly dilutedwith 20-30 volumes of a buffer containing 50 mM Tris-HCl, pH8 and 150 mMNaCl, adjusted to pH 6.6 and chromatographed with a strong cationexchange column (Poros HS-50). The protein was eluted with 3-5 CV of astepwise gradient of 300 mM, 700 mM, and 1500 mM NaCl in 50 mM MES at pH6.6. The fraction eluted with 0.7 M NaCl was diluted 3-fold with water,and applied to a set of strong anion (Poros HQ-50) and cation (PorosCM-20) exchange columns in a tandem mode. The CM column was eluted with10-20 CV of a linear gradient from 50 mM MES pH6.6, 150 mM NaCl to 50 mMTris-HCl pH 8, 500 mM NaCl. Fractions containing purified LIGHT asanalyzed by SDS-PAGE were combined.

Quality Control of the Recombinant Proteins

The endotoxin levels in the purified recombinant proteins weredetermined by the LAL assay on a Limulus Amebocyte Lysate (LAL)-5000Automatic Endotoxin Detection System (Associates of Cape Cod, Inc.Falmouth, Mass.), according to the standard procedure recommended by themanufacturer. All the recombinant proteins were subjected to N-terminalsequence using an ABI-494 sequencer (PE Biosystems, Inc. Foster City,Calif.) for their authenticity. The proteins was dialyzed against PBScontaining 20% (v/v) glycerol for storage at −80° C. For applicationssuch as CTL, cytokine secretion and heart transplantation, the proteinswere subsequently dialyzed against PBS to remove the glycerol in thesolution.

BIAcore Analysis

The binding of human LIGHT to human TR6-Fc was first assessed by BIAcoreanalysis (BIAcore Biosensor, Piscataway, N.J.). TR6-Fc or TR2-Fc fusionproteins were covalently immobilized to the BIAcore sensor chip (CM5chip) via amine groups usingN-ethyl-N′-(dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide.Various dilutions of LIGHT were passed through the TR6-Fc- orTR2-Fc-conjugated flow cells at 15 microliters/min for a total volume of50 microliters. The amount of bound protein was determined duringwashing of the flow cell with HBS buffer (10 mM HEPES, pH 7.4, 150 mMNaCl, 3.4 mM EDTA, 0.005% Surfactant P20). The flow cell surface wasregenerated by washing off the bound proteins with 20 microliters of 10mM glycine-HCl pH 2.3. For kinetic analysis the flow cells were testedat different flow rates and with different density of the conjugatedTR6-Fc or TR2-Fc proteins. The on- and off-rates were determinedaccording a kinetic evaluation program in the BiaEvaluation 3 softwareusing a 1:1 binding model and the global analysis method.

Generation of Stable Cell Lines that Express Human LIGHT

The full-length human LIGHT genes were PCR amplified and subcloned intopcDNA3.1. The parental vector and the LIGHT expression vectors were thentransfected into 293F cells (Life Technologies, Grand Island, N.Y.)using Lipofectamine (Life Technology) and stable clones resistant to 0.5mg/ml geneticin were selected.

Flow Cytometry

Cells were incubated with Fc-fusion proteins in 100 ul FACS buffer(d-PBS with 0.1% sodium azide and 0.1% BSA) for 15-20 minutes at roomtemperature. The cells were washed once and reacted with goat F (ab)₂anti-human IgG (Southern Biotechnology, Birmingham, Ala.) for 15 minutesat room temperature. After wash, the cells were resuspended in 0.5 ug/mlpropidium iodide, and live cells were gated and analyzed on a FACScan(BD Biosciences, Mansfield, Mass.).

Stimulation of Human T Cells for LIGHT Expression

Briefly, T cells were purified from human peripheral blood andstimulated with anti-CD3 in the presence of rhuIL-2 for 5 days. Thecells were restimulated with PMA (100 ng/ml) and ionomycin (1 mg/ml) foradditional 4 hours. LIGHT expression on the cells was assessed by thebinding of TR6-Fc (10 ng/sample), TR2-Fc (250 ng/sample) or Fas-Fc (250ng/sample) to the cells using flow cytometry.

Three-Way MLR of Human PBMC

PBMC from human donors were purified by density gradient usingLymphocyte Separation Medium (LSM, density at 1.0770 g/ml, OrganonTeknika Corporation, West Chester, Pa.). PBMC from three donors weremixed at a ratio of 2:2:0.2 for a final density of 4.2×10⁶ cells/ml inRPMI-1640 (Life Technologies) containing 10% FCS and 2 mM glutamine. Thecells were cultured for 5-6 days in round-bottomed microtitre plates(200 microliters/well) in triplicate, pulsed with [³H] thymidine for thelast 16 h of culture, and the thymidine uptake was measured as describebefore (Chen, H., et al., 1996. J. Immunol. 157:4297, which is herebyincorporated by reference in its entirety).

One-Way ex vivo MLR After in vivo Stimulation in Mice

The F1 of C57BL/6×BALB/c mice (H-2^(b×d)) were transfused i.v. with1.5×10⁸ spleen cells from C57BL/6 mice (H-2^(b)) on day 1. TR6-Fc or acontrol fusion protein was administered i.v. daily for 9 days at 3mg/kg/day starting one day before the transfusion. The spleen cells ofthe recipient F1 mice were harvested on day 8 for in vitro proliferationand cytokine assays.

Ex vivo Mouse Splenocyte Proliferation

Single splenocyte suspensions from normal and transfused F1 mice werecultured in triplicate in 96-well flat-bottomed plates (4×10⁵ cells/200microliters/well) for 2-5 days as with the human MLR. After removing 100microliters of supernatants per well on the day of harvest, 10microliters alamar Blue (Biosource, Camarillo, Calif.) was added to eachwell and the cells were cultured for additional 4 h. The cell number ineach well was assessed according to OD₅₉₀ using a CytoFlu apparatus(PerSeptive Biosystems, Framingham, Mass.).

Mouse Cytokine Assays

Cytokines in the culture supernatants of mouse spleen cells weremeasured with commercial ELISA kits from Endogen (Cambridge, Mass,) or R& D Systems (Minneapolis, Minn.).

Mouse Cytotoxic T Lymphocyte (CTL) Assay

Transgenic mice carrying L^(d)-specific TCR (2C mice) were used in thisexperiment. In the 2C mice, the majority (about 75%) of their T cellsare CD8⁺, and almost all the CD8⁺ cells carry clonotypic TCR recognizedby mAb 1B2. The 2C mice in our colony are of an H-2^(b) background. 2Cspleen cells were stimulated with an equal number of mitomycin C-treatedBALB/c spleen cells in 24-well plates at a final density of 4×10⁶cells/2 ml/well. After 5 days of culture in the presence of 10 U/mlrecombinant human IL-2, the viable cells were counted and assayed fortheir H-2^(d)-specific cytotoxic activity using ⁵¹Cr-labeled P815 cells(H-2^(d)) as targets. A standard 4-h ⁵¹Cr release assay (Chen, H., etal., 1996. J. Immunol. 157:4297, which is hereby incorporated byreference in its entirety) was carried out in 96-well round-bottomedplates with 0.15×10⁶ target cells/well/200 microliters at differentratios of effector/target cells (10:1, 3:1, 1:1 and 0.3:1). After 4-hincubation, 100 microliters of super was collected from each well andcounted in a gamma-counter. The percentage lysis of the test sample iscalculated as follows:

${\%\mspace{14mu}{lysis}} = \frac{{{cpm}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{test}\mspace{14mu}{sample}} - {{cpm}\mspace{14mu}{of}\mspace{14mu}{spontaneous}\mspace{14mu}{release}}}{{{cpm}\mspace{14mu}{of}\mspace{14mu}{maximal}\mspace{14mu}{release}} - {{cpm}\mspace{14mu}{of}\mspace{14mu}{spontaneous}\mspace{14mu}{release}}}$

where the spontaneous release is derived from 100 microliterssupernatant of the target cells cultured alone for 4 h, and the maximalrelease is derived from 100 microliters lysate of 0.15×10⁶ target cellswhich were lysed by SDS in a total volume of 200 microliters.

Mouse Heart Transplantation

Three- to four-month-old C57BL/6 mice (H-2^(b)) were used as recipients,and 2- to 3-month-old BALB/c mice (H-2^(d)) were used as donors. Theprocedure of heterotopic heart transplantation was detailed in Chen, H.,et al., 1996. J. Immunol. 157:4297, which is hereby incorporated byreference in its entirety. The contraction of the transplanted heart wasassessed daily by abdominal palpation. The duration between the day ofthe operation and the first day when a graft totally lost its palpableactivity was defined as the graft survival time. Animals that lostpalpable activity of the graft within three days after transplantationwere classified as technical failures (<5%) and were omitted from theanalysis.

Results of Example 23

Preparation of Recombinant Proteins of Human TR6-Fc, TR6, LIGHT, TR2-Fc,Fas-Fc and MCIF-Fc

The purified TR6-Fc protein was analyzed with SDS-PAGE under reducingand nonreducing conditions. The result demonstrate that the protein is adisulfide-linked dimer under the non-reducing condition. Lightscattering analysis also confirmed that the protein behaves as a dimerin solution. N-terminal sequencing revealed that the mature secretedTR6-Fc had the predicted sequence of VAETP starting at aa 30. Theestimated purity of the protein preparation was more than 98% accordingto SDS-PAGE. Endotoxin levels in the purified proteins were below 10EU/mg. Human TR6 without Fc, TR2-Fc, Fas-Fc and MCIF-Fc were alsoprepared to a similar purity as TR6-Fc and their authenticity wasverified with N-terminal sequencing.

The kinetics of Binding Between of TR6 and LIGHT

TR6-Fc has been previously shown to bind both LIGHT and FasL. Wedetermined the kinetics of binding of LIGHT to both the Fe and non-Fcversions of TR6 according to BIAcore analysis. The Kd for LIGHT bindingto TR6-Fc and non-Fc forms was 5.46 nM and 14.3 nM, respectively. Theoff rate (kd) for TR6 (4.83E-03 1/s) was approximately 2-fold higherthan that of TR6-Fc (2.30E-03 1/s). The on-rates, ka, were 4.22E05 and3.38E05 1/Ms for TR6-Fc and TR6, respectively, with TR6-Fc having aslightly higher on rate. The exact reason for the apparent higher Kdvalue for TR6-Fc compared to TR6-Fc is not known, but a comparabledifference in binding affinity was also observed with FasL. The bindingof LIGHT to TR2-Fc was also determined. The Kd was 4.56 nM, which isessentially the same as that between LIGHT and TR6-Fc.

TR6-Fc Binds LIGHT Directly and Can Compete with TR2 for the Binding ofLIGHT Overexpressed on 293 Cell Surface

After it was shown that TR6-Fc could bind to LIGHT in BIAcore chips, theability of TR6-Fc to bind to LIGHT expressed on cell surface wasanalyzed. This was tested on 293 cells overexpressing LIGHT according toflow cytometry. Fas-Fc was used as a control, and it did not bind to thetransfected cells. TR6-Fc could bind to the LIGHT-transfectants, but noton untransfected cells. The specificity of the binding was furtherdemonstrated by competition of TR6-Fc binding with soluble non-Fc formof TR6. Dose-dependent competition of TR6-Fc binding was attained usingincreasing concentrations of TR6 protein, and nearly complete inhibitionwas achieved with 10 micrograms of TR6.

It has been shown that TR2 can bind to LIGHT. Since TR6 also binds toLIGHT as shown above, its ability to interfere with the binding betweenTR2 and LIGHT was analyzed. This possibility was examined with flowcytometry. TR2-Fc could bind to the 293 cells overexpressing LIGHT asexpected. TR6 could compete off the binding in a dose-dependent fashion.At 10 micrograms of TR6, the binding of TR2-Fc to the 293 cells wasalmost completely disappeared.

The results from this section indicate that TR-6 can bind to the cellmembrane LIGHT, and it can also compete with TR2 for the binding ofLIGHT.

TR6-Fc Reactivity with Activated T Cells

LIGHT expression is upregulated on T cells activated with anti-CD3 andIL-2 followed by PMA and ionomycin treatment (Mauri, D. N., et al.,1998, Immunity. 8:21). Using this activation regimen, we confirmedprevious results according to flow cytometry that TR2-Fc bound to Tcells thus activated. We then extended this observation by showing thatas with TR2-Fc, TR6-Fc also bound to these activated T cells. Thebinding was specific because a control Fc fusion protein Fas-Fc did notbind to these cells, and the binding could be competed off with solubleTR6. The interaction between TR6 and the activated T cells was mediatedvia LIGHT expressed on these T cells, because the same soluble TR6protein could also compete off the binding of TR2-Fc and LTbetaR-Fc withthe T cells, TR2 and LTbetaR being receptors of LIGHT. These resultsdemonstrate that soluble TR6 could associate with endogenous LIGHTexpressed on the activated T cells, and it can interfere with theinteraction between LIGHT and TR2 in immune cells.

TR6-Fc Inhibits Human MLR

It has been shown that soluble LIGHT can enhance a 3-way MLR, andsoluble recombinant TR2-Fc can inhibit the 3-way MLR or dendriticcells-stimulated alloresponse of the T cells. These immune regulationsare likely via the interaction between soluble LIGHT and its cellsurface receptor TR2. Since TR6 could interfere with the interactionbetween LIGHT and TR2 as shown in our flow cytometry, we analzyed itsability to alter T cell alloresponses by testing the effect of TR6 in athree-way human MLR. The results show that TR6-Fc inhibited the T cellproliferation in this system. A control Fc fusion protein had no effect,whereas TR6-Fc at 1 microgram/ml caused nearly 50% inhibition. Furtherincrease of the TR6-Fc concentrations had no additional suppressiveeffect.

TR6-Fc Inhibits Splenocyte Alloactivation ex vivo in Mice

It has been shown previously that T cells stimulated by alloantigen invivo have increased spontaneous proliferation ex vivo, and alloreactiveT cells depend on LIGHT for some costimulation in certain case. Wetested whether TR6 had any immune regulatory effects in vivo onalloantigen-stimulated T cells. Parental splenocytes (H-2^(b)) weretransftised i.v. into H-2^(b×d) F1 mice, and the recipient mice weregiven TR6-Fc i.v. at 3 mg/kg/day for starting on day −1 (the day oftransfusion was designated as day 0). The F1 mice were sacrificed on day8 and the spleen weight of the mice were registered. The splenocyteswere then cultured without additional stimulation to measure theirspontaneous proliferation and cytokine production. Treatment with TR6-Fcreduced splenomagaly considerably, decreased spontaneous splenocyteproliferation as measured on day 4 after the culture, and inhibited theIFN-gamma and GM-CSF production by the spleen cells as measured from day2 to day 5 of the culture. In contrast, all mice treated with control Fcor buffer had significantly more severe splenomagaly, higher splenocyteproliferation and higher INF-gamma and GM-CSF productions. Thus, ourresults show that TR6-Fc is immunologically active and can indeedmodulate T cell-mediated alloactivation in vivo.

TR6-Fc and TR6 Inhibits Mouse CTL Activity Developed AgainstAlloantigens

L^(d)-specific transgenic 2C T cells were then used as a model system toevaluate the effect of TR6 on the differentiation ofalloantigen-specific CD8 cells into effector cells, since the CD8 cellsare mainly responsible to the alloresponsiveness, and the highalloreactive CD8 CTL precursors in the 2C mice gives out elevatedread-out signals for easy detection of possible changes exerted by TR6.In the presence of either TR6-Fc or TR6, the CTL activity was decreasedsignificantly compared with the cultures containing no recombinantprotein or containing normal human IgG. The detection of similar effectof TR6 and TR6-Fc in this experiment is of significant importance,because it excludes the possibility that the effect seen with TR6-Fc isFc-mediated. The CTL assay presented in the figure was carried out onday 6 of the culture. When CTL were assayed on day 5 of the culture,there was no obvious difference between samples with or without TR6.This indicates that the repression of CTL seen on day 6 is not due to akinetic shift.

TR6-Fc Modulates Lymphokine Production of2C T Cells Stimulated withH-2^(d) Alloantigens in vitro

The CTL differentiation and maturation are modulated by a plethora oflymphokines, and we examined the production of battery of lymphokinesproduced by 2C spleen cells upon stimulation of mitomycin C-treatedBALB/c spleen cells (H-2^(d)) in the presence of TR6-Fc. There was asuppression of IL-2 production between 24-72 h after the stimulation,while the levels of IL-10 were upregulated.

TR6-Fc Prolongs Heart Allograft Survival of the Mice

Since TR6-Fc could repress ex vivo T cell proliferation after thealloantigen stimulation, and inhibit CTL development in in vitro assays,we speculated that it could also modulate a more complete immuneresponse such as graft rejection. This was tested in a model of mouseheterotopic heart allografting, with C57BL/6 as recipients and BALB/c asdonors. The recipients were administrated with TR6-Fc i.v. daily at 7.5mg/kg/day for 7 days starting from one day before the operation. Forthis test group, the mean survival time (MST) of the grafts was 10.0+1.2days, while the MST of the control group was 6.8+0.4 days. Thedifference between the two groups was highly significant (p=0.0001,non-paired Student's t test). This result shows that TR6-Fc couldmodulate an authentic immune response such as allograft rejection.

Example 24 TNFR-6 alpha, TNFR-6 beta and DR3 Interact withTNF-Gamma-beta

The premyeloid cell line TF-1 was stably transfected with SRE/SEAP(Signal Response Element/Secreted Alkaline Phosphatase) reporter plasmidthat responds to the SRE signal transduction pathway. The TF1/SREreporter cells were treated with TNF-gamma-beta (InternationalPublication Numbers WO96/14328, WO00/66608, and WO00/08139) at 200 ng/mLand showed activation response as recorded by the SEAP activity. Thisactivity can be neutralized by A TNFR-6 alpha Fc fusion protein(hereinafter TR6.Fc in this example) in a dose dependent manner. TheTR6.Fc by itself, in contrast, showed no activity on the TFI/SREreporter cells. The results demonstrate that 1) TF-1 is a target cellfor TNF-gamma-beta ligand activity; and 2) TR6 interacts withTNF-gamma-beta and inhibits its activity on TF-1 cells. TR6 is known tohave two splice forms, TR6-alpha and and TR6-beta; both splice formshave been shown to interact with TNF-gamma-beta.

Similarly, the interaction of DR3 (International Publication NumbersWO97/33904 and WO/0064465) and TNF-gamma-beta can be demonstrated usingTF-1/SRE reporter cells. The results indicate that DR3.Fc interacts withTNF-gamma-beta, either by competing naturally expressed DR3 on TF-1cells or forming inactive TNF-gamma-beta /DR3.fc complex, or both.

At least three additional pieces of evidence demonstrate an interactionbetween TNF-gamma-beta and DR3 and TR6. First, both TR6.Fc and DR3.Fcare able to inhibit TNF-gamma-beta activation of NFkB in 293T cells,whereas in the same experiment, TNFR1.Fc was not able to inhibitTNF-gamma-beta activation of NFKB in 293T cells. Secondly, both TR6.Fcand DR3.Fc can be used to immunoprecipitate TNF-gamma-beta. Thirdly,TR6.Fc proteins can be detected by FACS analysis to specifically bindcells transfected with TNF-gamma-beta.

Example 25 TNF-gamma-beta is a Novel Ligand for DR3 and TR6-alpha (DcR3)and Functions as a T Cell Costimulator

Introduction

Members of the TNF and TNFR superfamilies of proteins are involved inthe regulation of many important biological processes, includingdevelopment, organogenesis, innate and adaptive immunity (Locksley etal., Cell 104:487-501 (2001)). Interaction of TNF ligands such as TNF,Fas, LIGHT and BLyS with their cognate receptor (or receptors) has beenshown to affect the immune responses, as they are able to activatesignaling pathways that link them to the regulation of inflammation,apoptosis, homeostasis, host defense, and autoimmunity. The TNFRsuperfamily can be divided into two groups based on the presence ofdifferent domains in the intracellular portion of the receptor. Onegroup contains a TRAF binding domain that enables them to couple toTRAFs (TNFR-associated factor); these in turn activate a signalingcascade that results in the activation of NF-κB and initiation oftranscription. The other group of receptors is characterized by a 60amino acid globular structure named Death Domain (DD). Historicallydeath domain-containing receptors have been described as inducers ofapoptosis via the activation of caspases. These receptors include TNFR1,DR3, DR4, DR5, DR6 and Fas. More recent evidence (Siegel et al., NatureImmunology 1:469-474 (2000) and references within) has shown that somemembers of this subgroup of receptors, such as Fas, also have theability to positively affect T cell activation. A third group ofreceptors has also been described. The members of this group, thatinclude DcR1, DcR2, OPG, and TNFR-6 alpha (also called DcR3, andhereinafter in this example referred to as “TR6”), have been named decoyreceptors, as they lack a cytoplasmic domain and may act as inhibitorsby competing with the signal transducing receptor for the ligand(Ashkenazi et al., Curr. Opin. Cell Biol. 11:255-260 (1999)). TR6, whichexhibits closest homology to OPG, associates with high affinity to FasLand LIGHT, and inhibits FasL-induced apoptosis both in vitro and in vivo(Pitti et al., Nature 396:699-703 (1998), Yu, et al., J. Biol. Chem.274:13733-6 (1999); Connolly, et al., J. Pharmacol. Exp. Ther. 298:25-33(2001)). Its role in down-regulating immune responses was stronglysuggested by the observation that TR6 surpresses T-cell responsesagainst alloantigen (Zhang et al., J. Clin. Invest. 107:1459-68 (2001))and certain tumors overexpress TR6 (Pitti et al., Nature 396:699-703(1998), Bai et al., Proc. natl. Acad. Sci. 97:1230-1235 (2000)).

DR3 (described in International Publication Numbers WO97/33904 andWO/0064465 which are herein incorporated by reference in theirentireties) is a DD-containing receptor that shows highest homology toTNFR1 (Chinnaiyan et al., Science 274:990-2 (1996); Kitson et al.,Nature 384:372-5 (1996), Marsters et al., Curr. Biol. 6:1669-76 (1996);Bodmer et al., Immunity 6:79-88 (1997); Screaton et al., Proc. Natl.Acad. Sci. 94:4615-19 (1997); Tan et al., Gene 204:35-46 (1997)). Incontrast to TNFR1, which is ubiquitously expressed, DR3 appears to bemostly expressed by lymphocytes and is efficiently induced following Tcell activation. TWEAK/Apo3L was previously shown to bind DR3 in vitro(Marsters et al., Curr. Biol. 8:525-528 (1998)). However, more recentwork raised doubt about this interaction and showed that TWEAK was ableto induce NF-κB and caspase activation in cells lacking DR3 (Schneideret al., Eur. J. Immunol. 29:1785-92 (1999); Kaptein et al., FEBS Letters485:135-141 (2000)).

In this example, the characterization of the ligand, TNF-gamma-beta(also known as TL1β; described in International Publication Numbers:WO00/08139 and WO00/66608 which are herein incorporated by reference intheir entireties), for both DR3 and TR6/DcR3 is described.TNF-gamma-beta is a longer variant of TNF-gamma-alpha (also known asVEGI and TL1; described in International Publication Numbers WO96/14328,WO99/23105, WO00/08139 and WO00/66608 which are herein incorporated byreference in their entireties), which was previously identified as anendothelial-derived factor that inhibited endothelial cell growth invitro and tumor progression in vivo (Tan et al., Gene 204:35-46 (1997);Zhai et al., FASEB J. 13:181-9 (1999); Zhai et al., Int. J. Cancer82:131-6 (1999); Yue et al., J. Biol. Chem. 274:1479-86 (1999)). It wasfound that TNF-gamma-beta is the more abundant form than TNF-gamma-alphaand is upregulated by TNFα and IL-1α. U.S. Pat. No. 5,876,969

As shown herein, the interaction between TNF-gamma-beta and DR3 in 293Tcells and in the erythroleukemic line TF-1 results in activation ofNF-κB and induction of caspase activity, respectively. TR6 is able toinhibit these activities by competing with DR3 for TNF-gamma-beta. Moreimportantly, it was found that in vitro, TNF-gamma-beta functionsspecifically on activated T cells to promote survival and secretion ofthe proinflammatory cytokines IFNγ and GMCSF, and it markedly enhancesacute graft-versus-host reactions in mice.

Results

TNF-gamma-beta is a Longer Variant of TNF-gamma-alpha, a Member of theTNF Superfamily of Ligands

To identify novel TNF like molecules, a database of over three millionhuman expressed sequence tag (EST) sequences was analyzed using theBLAST algorithm. Several EST clones with high homology to TNF likemolecule 1, TNF-gamma-alpha (Tan et al., Gene 204:35-46 (1997); Zhai etal., FASEB J. 13:181-9 (1999) Yue et al., J. Biol. Chem 274:1479-86(1999)) were identified from endothelial cell cDNA libraries. Sequenceanalysis of these cDNA clones revealed a 2080 base pair (bp) insertencoding an open reading frame of 251 amino acids (aa) with two upstreamin-frame stop codons. The predicted protein lacks a leader sequence butcontains a hydrophobic transmembrane domain near the N-terminus, and acarboxyl domain that shares 20-30% sequence similarity with other TNFfamily members. Interestingly, the C-terminal 151-aa of this protein(residues 101-251) is identical to residues 24 to 174 ofTNF-gamma-alpha, whereas the amino-terminal region shares no sequencesimilarity. The predicted extracellular receptor-interaction domain ofTNF-gamma-beta contains two potential N-linked glycosylation sites andshows highest amino acid sequence identity to TNF (24.6%), followed byFasL (22.9%) and LTα (22.2%). A 337-bp stretch of the TNF-gamma-betacDNA, containing most of the 5′ untranslated region and the sequencesencoding the first 70 amino acids of the TNF-gamma-beta protein, matchesa genomic clone on human chromosome 9 (Genbank Accession: AL390240,clone RP11-428F18). Further analysis of the human genomic sequencesreveals that TNF-gamma-alpha and TNF-gamma-beta are likely derived fromthe same gene. While TNF-gamma-beta is encoded by four putative exons,similar to most TNF-like molecules, TNF-gamma-alpha is encoded by onlythe last exon and the extended N-terminal intron region, and thereforelacks a putative transmembrane domain and the first conserved β-sheet

Mouse and rat TNF-gamma-beta cDNAs isolated from normal kidney cDNAseach encode a 252-aa protein. The overall amino acid sequence homologybetween human and mouse, and human and rat TNF-gamma-beta proteins is63.7% and 66.1%, respectively. Higher sequence homology was found in thepredicted extracellular receptor-interaction domains, of which human andmouse share 71.8% and human and rat share 75.1% sequence identity. An84.2% sequence identity is seen between the mouse and rat TNF-gamma-betaproteins.

Like most TNF ligands, TNF-gamma-beta exists as a membrane-bound proteinand can also be processed into a soluble form when ectopicallyexpressed. The N-terminal sequence of soluble TNF-gamma-beta proteinpurified from full length TNF-gamma-beta transfected 293T cells wasdetermined to be Leu 72.

TNF-gamma-beta is Predominantly Expressed by Endothelial Cells, a MoreAbundant Form than TNF-gamma-alpha, and is Inducible by TNF and IL-1α

To determine the expression pattern of TNF-gamma-beta, TNF-gamma-betaspecific primer and fluorescent probe were used for quantitativereal-time polymerase chain reaction (TaqMan) and reverse transcriptasepolymerase chain reaction (RT-PCR) (see Experimental Procedures below).TNF-gamma-beta is expressed predominantly by human endothelial cells,including the umbilical vein endothelial cells (HUVEC), the adult dermalmicrovascular endothelial cells (HMVEC-Ad), and uterus myometrialendothelial cells (UtMEC-Myo), with highest expression seen in HUVEC. A˜750 bp DNA fragment was readily amplified from these endothelial cellsby RT-PCR, indicating the presence of full length TNF-gamma-betatranscripts. Very little expression was seen in human aortic endothelialcells (HAEC) or other human primary cells including adult dermalfibroblast (NHDF-Ad and HFL-1), aortic smooth muscle cells (AoSMC),skeletal muscle cells (SkMC), adult keratinocytes (NHEK-Ad), tonsillar Bcells, T cells, NK cells, monocytes, or dendritic cells. Consistent withthese results, TNF-gamma-beta RNA was detected in human kidney,prostate, stomach, and low levels were seen in intestine, lung, andthymus, but not in heart, brain, liver, spleen, or adrenal gland. Nosignificant levels of TNF-gamma-beta mRNA in any of the cancer celllines tested, including 293T, HeLa, Jurkat, Molt4, Raji, IM9, U937,Caco-2, SK-N-MC, HepG2, KS4-1, and GH4C were detected.

As the expression pattern of TNF-gamma-beta is very similar to that ofTNF-gamma-alpha (Tan et al., Gene 204:35-46 (1997); Zhai et al., FASEBJ. 13:181-9 (1999)), the relative abundance of the two RNA species wasanalyzed using TNF-gamma-alpha and TNF-gamma-beta specific primers andfluorescence probes for conventional and quantitative RT-PCR. MoreTNF-gamma-beta mRNA was detected than that of TNF-gamma-alpha using bothmethods. The amount of TNF-gamma-beta mRNA is at least 15-fold higherthan that of TNF-gamma-alpha in the same RNA samples. To determine ifTNF-gamma-beta mRNA levels were inducible, HUVEC cells were stimulatedwith either TNF, IL-1α, PMA, bFGF or IFNγ. PMA and IL-1α rapidly inducedhigh levels of TNF-gamma-beta mRNA, with a peak in expression reached at6 hours after treatment. TNF was also able to induce TNF-gamma-betamRNA, but the time course of induction appeared to be delayed comparedto PMA and IL-1α. In contrast, bFGF and IFNγ did not significantlyaffect the expression of TNF-gamma-beta. TNF-gamma-beta protein levelsin the supernatants of activated HUVEC cells were analyzed by ELISA anda similar profile of induction was observed.

Identification of DR3 and TR6 as Receptors for TL1β

To identify the receptor for TNF-gamma-beta, we generated HEK293F stabletransfectants expressing full length TNF-gamma-beta on the cell surface(confirmed by Taqman and flow cytometric analysis using TNF-gamma-betamonoclonal antibody). These cells were used to screen the Fc-fusion formof the extracellular domain of TNFR family members, including TNFR1,Fas, HveA, DR3, DR4, DR5, DR6, DcR1, DcR2, TR6,OPG, RANK,AITR, TACI,CD40, and OX40. DR3-Fc and TR6-Fc bound efficiently to cells expressingTNF-gamma-beta but not to vector control transfected cells. In contrast,HveA-Fc and all the other receptors tested did not bind to theTNF-gamma-beta expressing cells. TR6 has been previously described as adecoy receptor (Pitti et al., Nature 396:699-703 (1998); Yu et al., J.Biol Chem. 274:13733-6 (1999)) capable of competing with Fas and HveAfor binding of FasL and LIGHT, respectively. Whether TR6 could competewith DR3 for TNF-gamma-beta binding was tested. When a 2:1 molar ratioof a non-tagged form of TR6 and DR3-Fc were used, no binding of DR3-Fcwas detected on TNF-gamma-beta expressing cells. These resultsdemonstrated that both DR3 and TR6 can bind to membrane-bound form ofthe TNF-gamma-beta protein.

Whether TNF-gamma-beta protein could bind to membrane-bound form of thereceptor, DR3 was tested. A FLAG-tagged soluble form of the TL1β. (aa72-251) protein was tested for binding of cells transiently transfectedwith different members of the TNFR family, including TNFR2, LTβ R,4-1BB, CD27, CD30, BCMA, DR3, DR4, DR5, DR6, DcR1, DcR2, RANK, HveA, andAITR. Binding of FLAG-TL1β, to cells expressing full length orDD-deleted DR3, but not to any of the other receptors tested, wasconsisitently detected, demonstrating that TNF-gamma-beta interacts withmembrane-associated DR3. The small shift (˜30%) seen when full lengthDR3 was used is likely due to the presence of low DR3-expressing cellswhile DR3 overexpressed cells undergone apoptosis.

Coimmunoprecipitation studies were also performed to confirm thatTNF-gamma-beta could specifically bind DR3 and TR6. Consistent with whatwe observed in FACS analysis, we found that DR3-Fc and TR6-Fcspecifically interacted with FLAG-TNF-gamma-beta. In contrast, Fas-Fc orTACI-Fc could not immunoprecipitate FLAG-TNF-gamma-beta, but efficientlybound their known ligands, FLAG-FasL and FLAG-BlyS, respectively.

To verify that the TNF-gamma-beta binding to DR3 and TR6 was specificand exhibited characteristics that were similar to those observed withother TNF family members to their cognate receptors, a BIAcore analysisusing a non-tagged TNF-gamma-beta (aa 72-251) protein purified from E.coli was perfomed. The kinetics of TNF-gamma-beta binding to DR3-Fc wasdetermined using three different batches of the TNF-gamma-beta protein.The ka and kd values were found to be 6.39E+05 Ms⁻¹ and 4.13E-03M⁻¹,respectively. The average Kd value was 6.45±0.2 nM. TNF-gamma-beta wasalso examined for its ability to bind to several other TNF-relatedreceptors (HveA, BCMA, TACI, and TR6). In addition to DR3, only TR6 wasfound to have significant and specific binding to TNF-gamma-beta. The kaand kd values were 1.04E+06 Ms⁻¹ and 1.9E-03 M⁻¹ respectively, whichgives a Kd of 1.8 nM. The specificity of binding of TL1β to DR3-Fc andTR6-Fc were confirmed by the competition of TNF-gamma-beta binding inthe presence of excess soluble receptor-Fc. These Kd values for bindingof TNF-gamma-beta to DR3-Fc and TR6-Fc are comparable to thosedetermined for other TNFR-ligand interactions.

Interaction of TL1β with DR3 Induces Activation of NF-κB

Previous reports have demonstrated that ectopic expression of DR3results in the activation of the transcription factor NF-κB (Chinnaiyanet al., Science 274:990-2 (1996); Kitson et al., Nature 384:372-5(1996), Marsters et al., Curr. Biol. 6:1669-76 (1996); Bodmer et al.,Immunity 6:79-88 (1997)). TNF-gamma-beta induced signaling in areconstituted system in 293T cells in which DR3 and a NF-κB-SEAPreporter were introduced by transient transfection was studied. To avoidspontaneous apoptosis or NF-κB activation accompanied with DR3overexpression, a limited amount of DR3-expression DNA that by itselfminimally activated these pathways was used. Under these conditions,cotransfection of cDNAs encoding full length or the soluble form ofTNF-gamma-beta resulted in significant NF-κB activation. This signalingevent was dependent on the ectopic expression of DR3 and the intactnessof the DR3 death domain, as TNF-gamma-beta alone or in combination witha DD-deleted DR3 did not induce NF-κB activation in these cells.Cotransfection of DR3 with cDNAs encoding TNF-gamma-alpha (full lengthor N-terminal 24-aa truncated) failed to induce NF-κB activation. Asimilar induction of NF-κB activity was observed when increasing amountsof recombinant TL1β protein (aa 72-251, with or without FLAG® tag) wereadded to DR3 expressing cells. This induction of NF-κB was specificallyinhibited by the addition of excess amount of DR3-Fc or TR6-Fc, but notby the addition of Fas-Fc or TNFR1-Fc. These results demonstrated thatTNF-gamma-beta is a signaling ligand for DR3 that induces NF-κBactivation, and TR6 can specifically inhibit this event.

TL1β Induces IL-2 Responsiveness and Cytokine Secretion from Activated TCells

As DR3 expression is mostly restricted to the lymphocytes (Chinnaiyan etal., Science 274:990-2 (1996); Kitson et al., Nature 384:372-5 (1996);Marsters et al., Curr. Biol. 6:1669-76 (1996); Bodmeret al., Immunity6:79-88 (1997); Screaton et al., Proc. Natl. Acad. Sci. 94:4615-19(1997); Tan et al., Gene 204:35-46 (1997)) and is upregulated upon Tcell activation, we examined the biological activity of TNF-gamma-betaon T cells. Recombinant TNF-gamma-beta (aa 72-251) protein was testedfor its ability to induce proliferation of resting or costimulated Tcells (treated with amounts of anti-CD3 and anti-CD28 that are notsufficient to induce proliferation). In resting or costimulated T cells,no significant increase in proliferation over background was observed.Interestingly, cells that were previously treated with TNF-gamma-betafor 72 hours were able to proliferate significantly in the presence ofIL-2 than cells without TNF-gamma-beta preincubation, indicating thatTNF-gamma-beta increases the IL-2 responsiveness of costimulated Tcells.

As enhanced IL-2 responsiveness has been associated with increased IL-2receptor expression and altered cytokine secretion, it was of interestto assess these responses on costimulated T cells treated withTNF-gamma-beta. TNF-gamma-beta treatment indeed upregulated IL-2Rα(CD25) and IL-2Rβ (CD122) expression from these cells. The extent of theincrease in IL-2 receptor expression is consistent with the moderateincrease in IL-2 responsiveness compared with IL-2 itself. We nextmeasured cytokine secretion from these cells and found that both IFNγand GMCSF were significantly induced, whereas IL-2, IL-4, IL-10, or TNFwere not. This effect was mostly dependent on the T cell coactivatorCD28, as treatment of the cells with anti-CD3 and TNF-gamma-beta onlyminimally induced cytokine secretion. The effect that we observed on Tcells was specifically mediated by TNF-gamma-beta, as addition ofmonoclonal neutralizing antibody to TL1β, or addition of DR3-Fc orTR6-Fc proteins was able to inhibit TNF-gamma-beta-mediated IFNγsecretion. TNF-gamma-beta was also tested on a variety of primary cells,including B cells, NK cells, and monocytes, but no significant activitywas detected, suggesting a specific activity of TNF-gamma-beta on Tcells.

TL1β Induces Caspase Activation in TF-1 Cells but not in T Cells

Overexpression of DR3 in cell lines induces capase activation(Chinnaiyan et al., Science 274:990-2 (1996); Kitson et al., Nature384:372-5 (1996); Marsters et al., Curr. Biol. 6:1669-76 (1996); Bodmeret al., Immunity 6:79-88 (1997)). We tested whether TL1β could inducecaspase activation in primary T cells. Purified T cells were activatedwith PHA and incubated with recombinant TNF-gamma-beta or FasL in thepresence or absence of cycloheximide (CHX). No induction of caspaseactivity was detected in TNF-gamma-beta treated T cells, but was readilymeasured when cells were triggered with FasL, suggesting that under thisexperimental condition, TNF-gamma-beta does not activate caspases in Tcells (the assay we used detects activation of caspases 2, 3, 6, 7, 8,9, and 10). Various cell lines for the expression of DR3 and found thatthe erythroleukimic cell line TF-1 expressed high levels of DR3 werethen analyzed. The effect of recombinant TNF-gamma-beta protein oncaspase activation in TF-1 cells was then measured. In the absence ofcycloheximide, no significant increase in caspase activity was detectedfollowing TNF-gamma-beta treatment, while TNF-gamma-beta was able toefficiently induce caspase activation in the presence of cycloheximide.This effect was inhibited by either DR3-Fc or TR6-Fc protein but not byLIGHT-Fc. An anti-TNF-gamma-beta monoclonal antibody was also shown tocompletely inhibit this activity, confirming that the caspase activationwas mediated by TNF-gamma-beta.

TL1β Promotes Splenocyte Alloactivation in Mice

To determine if the in vitro activities of TNF-gamma-beta could bereproduced in vivo, a mouse model of acute graft-versus-host-response(GVHR) was developed in which parental C57BL/6 splenocytes were injectedintravenously into (BALB/c×C57 BL/6) F1 mice (CB6F1), and therecipient's immune responses were measured. Typical alloactivationresults in increased splenic weight of the recipient mice and enhancedproliferation and cytokine production of the splenocytes culturedex-vivo (Via, J. Immunol. 146:2603-9 (1991); Zhang et al., J. Clin.Invest. 107:1459-68 (2001)). The large number of T cells in the spleenand their expected upregulation of DR3 in response to alloactivationmakes this an ideal model to assess the effect of TNF-gamma-beta on adefined in vivo immune response. Five day administration of 3 mg/kg ofthe recombinant TNF-gamma-beta protein markedly enhanced thegraft-versus-host responses. The mean (n=4) weight of normal spleensobtained from naive CB6F1 mice was 0.091 g. Alloactivation resulted in a2.5 fold increase in splenic weight (˜228 g). Treatment of allograftedCB6F1 mice with recombinant TNF-gamma-beta protein (aa 72-251) furtherincreased splenic weight about 50%, to a mean value of 0.349 g.TNF-gamma-beta treatment also significantly enhanced ex-vivo splenocyteexpansion, and secretion of IFNγ and GMCSF. Thus, TNF-gamma-betastrongly enhances GVHR in vivo, and this effect is consistent withTNF-gamma-beta's in vitro activities.

Experimental Procedures

Cells, Constructs, and Other Reagents

All human cancer cell lines and normal lung fibroblast (HFL-1) werepurchased from American Tissue Culture Collection. Human primary cellswere purchased from Clonetics Corp. Cells were cultured as recommended.Human cDNA encoding the full length TNF-gamma-alpha, TNF-gamma-beta,DR3; the extracellular domain of TNF-gamma-alpha (aa 25-174),TNF-gamma-beta (aa 72-251), BlyS (aa 134-285), FasL (aa 130-281), anddeath domain truncated DR3 (DR3ΔDD, aa 1-345) were amplified by PCR andcloned into the mammalian expression vectors pC4 and/or pFLAG-CMV™1(Sigma). The extracellular domain of human DR3 (aa 1-199), TACI (aa 1-159), HveA (aa 1-192), Fas (aa 1-169), and full length TR6 (aa 1-300),was each fused in-frame, at its C-terminus, to the Fc domain of humanIgG1 and cloned into pC4. Rabbit polyclonal TNF-gamma-beta antibody wasgenerated using recombinant TNF-gamma-beta (aa 72-251) protein andpurified on a TNF-gamma-beta affinity column. Monoclonal antibodies wereraised against recombinant TNF-gamma-beta as described (Kohler andMilstein, Nature 256:503-519 (1975)).

Cloning of Human, Mouse, and Rat TNF-gamma-beta cDNA

TNF-gamma-beta was identified by screening a human EST database forsequence homology with the extracellular domain of TNF, using the blastnand tblastn algorithms. The extracellular domain of the mouse and ratTNF-gamma-beta cDNA was isolated by PCR amplification from mouse or ratkidney Marathon-Ready cDNAs (Clontech) using human TNF-gamma-betaspecific primers. The resulting sequences were then used to design mouseand rat TNF-gamma-beta specific primers to amplify the 5′ and 3′ ends ofthe cDNA using Marathon cDNA Amplification kit (Clontech). Each sequencewas derived and confirmed from at least two independent PCR products.

Generation of TNF-gamma-beta Stable Cell Line

HEK293F cells were transiently transfected with pcDNA3.1(+) (vectorcontrol) or pcDNA3.1(+) containing full length TNF-gamma-beta. Cellsresistant to 0.5 mg/ml Genticin (Invitrogen) were selected and expanded.Expression of TNF-gamma-beta mRNA was confirmed by quantitative RT-PCRanalysis and surface expression of TNF-gamma-beta protein confirmed byFACS analyses using TNF-gamma-beta monoclonal antibodies.

Quantitative Real-Time PCR (TaqMan) and RT-PCR Analysis

Total RNA was isolated from human cell lines and primary cells usingTriZOL (Invitrogen). TaqMan was carried out in a 25 microliter reactioncontaining 25 ng of total RNA, 0.6 μM each of gene-specific forward andreverse primers and 0.2 μM of gene-specific fluorescence probe.TNF-gamma-beta specific primers (forward:5′-CACCTCTTAGAGCAGACGGAGATAA-3′ (SEQ ID NO:34), reverse:5′-TTAAAGTGCTGTGTGGGAGTTTGT-3′ (SEQ ID NO:35), and probe:5′-CCAAGGGCACACCTGACAGTTGTGA-3′ (SEQ ID NO:36)) amplify an amplicon spannucleotide 257 to 340 of the TNF-gamma-beta cDNA (aa 86-114 of theprotein), while TNF-gamma-alpha specific primers (forward:5′-CAAAGTCTACAGTTTCCCAATGAGAA-3′ (SEQ ID NO:37); reverse:5′-GGGAACTGATTTTTAAAGTGCTGTGT-3′ (SEQ ID NO:38); probe:5′-TCCTCTTTCTTGTCTTTCCAGTTGTGAGACAAAC-3′ (SEQ ID NO:39)) amplifynucleotide 17 to 113 of the TNF-gamma-alpha cDNA (aa 7-37 of theprotein). Gene-specific PCR products were measured using an ABI PRISM7700 Sequence Detection System following the manufacturer's instruction(PE Corp.). The relative mRNA level of TNF-gamma-beta was normalized tothe 18S ribosomal RNA internal control in the same sample.

For RT-PCR analysis, 0.5 micrograms of total RNA was amplified withTNF-gamma-alpha (5′-GCAAAGTCTACAGTTTCCCAATGAGAAAATTAATCC-3′(SEQ IDNO:40)) or TNF-gamma-beta specific sense primer(5′-ATGGCCGAGGATCTGGGACTGAGC-3′ (SEQ ID NO:41)) and an antisense primer(5′-CTATAGTAAGAAGGCTCCAAAGAAGGTTTTATCTTC-3′ (SEQ ID NO:42)) usingSuperScript One-Step RT-PCR System (Invitrogen). β-actin was used asinternal control.

Transfection and NF-κB Reporter Assay

293T cells were transiently transfected using LipofectAMINE and PLUSreagents according to the manufacturer's instruction (Invitrogen). Forreporter assays, 293T cells, at 5×10⁵ cells/well, were seeded in 6-wellplates and transfected with a total of 1 microgram of DNA. pC4 DNA wasused as filler DNA. Conditioned supernatant was collected 24 hrpost-transfection and assayed for secreted alkaline phosphatase (SEAP)activity using the Phospha-Light™ chemiluminescent reporter gene assaysystem (Tropix). pCMV-lacZ was used as internal control for transfectionefficiency normalization.

Recombinant Protein Purification

FLAG fusion proteins were produced from 293T cells by transienttransfection, and purified on anti-FLAG® M2 affinity columns (Sigma)according to manufacturer's instruction. Receptor proteins with orwithout Fc fusion were produced from Baculovirus or CHO stable celllines as described (Zhang et al., J. Clin. Invest. 107:1459-68 (2001)).Recombinant, untagged TNF-gamma-beta protein (aa 72-251) was generatedand purified from E. coli. Briefly, E. Coli cell extract was separatedon a HQ-50 anion exchange column (Applied Biosystems) and eluted with asalt gradient. The 0.2 M NaCl elution was diluted and loaded on a HQ-50column, and the flow through was collected, adjusted to 0.8 M ammoniasulfate and loaded on a Butyl-650s column (Toso Haus). The column waseluted with a 0.6M to 0 M ammonia sulfate gradient and the fractionscontaining TNF-gamma-beta protein were pooled and further purified bysize exclusion on a Superdex-200 column (Pharmacia) in PBS. Allrecombinant proteins were confirmed by NH₂-terminal sequencing on aABI-494 sequencer (Applied Biosystem). The endotoxin level of thepurified protein was less than 10 EU/mg as measured on a LAL-5000E (CapeCod Associates).

Flow Cytometry, Immunoprecipitation, and Western Blot Analysis

One million cells, in 0.1 ml of FACS buffer (PBS, 0.1% BSA, 0.1% NaN₃),were incubated with 0.1-1 microgram of protein or antibody at RT for 15min. The cells were washed with 3 ml of FACS buffer, reacted withbiotinylated primary antibody, and stained with PE-conjugated secondaryantibody at RT for 15 min. Cells were then washed again, resuspended in0.5 microgram/ml of propidium iodide, and live cells were gated andanalyzed on a FACScan using the CellQuest software (BD Biosciences).

For coimmunoprecipiation studies, 2 micrograms each of purified TNFR-Fcproteins was incubated with 1 microgram of Flag-tagged TNF-gamma-beta,FasL or BlyS protein and 20 microliters of protein A-Sepharose beads in0.5 ml of IP buffer (DMEM, 10% FCS, 0.1% Triton X-100) at 4° C for 4 hr.The beads then precipitated and washed extensively with PBST buffer(PBS, 0.5% Triton X-100) before boiled in SDS-sample buffer. Proteinswere resolved on 4-20% Tris-Glycine gels (NOVEX), transferred tonitrocellulose membranes, and blotted with anti-FLAG® M2 monoclonalantibody (1 microgram/ml, Sigma) andhorseradish peroxidase(HRP)-conjugated goat anti-mouse IgG antibody (0.5 microgram/ml).

BIAcore Analysis

Recombinant TNF-gamma-beta (from E. Coli) binding to various human TNFreceptors was analyzed on a BIAcore 3000 instrument. TNFR-Fc werecovalently immobilized to the BIAcore sensor chip (CM5 chip) via aminegroups usingN-ethyl-N′-(dimethylaminopropyl)carbodiimide/N-hydroxysuccinimidechemistry. A control receptor surface of identical density was prepared,BCMA-Fc, that was negative for TNF-gamma-beta binding and used forbackground subtraction. Eight different concentrations of TNF-gamma-beta(range: 3-370 nM) were flowed over the receptor-derivatized flow cellsat 15 microliters/min for a total volume of 50 microliters. The amountof bound protein was determined during washing of the flow cell with HBSbuffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% SurfactantP20). The flow cell surface was regenerated by displacing bound proteinby washing with 20 microliters of 10 mM glycine-HCl, pH 2.3. For kineticanalysis, the on and off rates were determined using the kineticevaluation program in BIAevaluation 3 software using a 1:1 binding modeland the global analysis method.

T cell Proliferation Assays.

Whole blood from human donors was separated by Ficoll (ICNBiotechnologies) gradient centrifugation and cells were culturedovernight in RPMI containing 10% FCS (Biofluids). T cells were separatedusing the MACS PanT separation kit (Milteny Biotech), the T cell purityachieved was usually higher that 90%. The cells were seeded on anti-CD3(0.3 microgram/ml, Pharmingen) and anti-CD28 (5.0 microgram/ml) coated96-well plates at 2×10⁴/well, and were incubated with medium alone, 1ng/ml of IL-2 (R & D Systems), or 100 ng/ml of TNF-gamma-beta (aa72-251) at 37° C. After 72 hour in culture, the cells were eitheruntreated or treated with 1 ng/ml of IL-2, and pulsed with 0.5 μCi of³H-thymidine for another 24 hours and incorporation of ³H measured on ascintillation counter.

Cytokine ELISA Assays for Primary Cells

1×10⁵ cells/ml of purified T cells were seeded in a 24-well tissueculture plate that had been coated with anti-CD3 (0.3 microgram/ml) andanti-CD28 (5.0 microgram/ml) overnight at 4° C. RecombinantTNF-gamma-beta (aa72-251) protein (100 ng/ml) was added to cells andsupernatants were collected 72 hours later. ELISA assay for IFNγ,GM-CSF, IL-2 IL-4, IL-10 and TNFα were performed using kits purchasedfrom R & D Systems. Recombinant human IL-2 (5 ng/ml) was used as apositive control. All samples were tested in duplicate and results wereexpressed as an average of duplicate samples plus or minus error.

Caspase Assay

TF-1 cells or PHA-activated primary T cells were seeded at 75,000cells/well in a black 96-well plate with clear bottom (Becton Dickinson)in RPMI Medium containing 1% fetal bovine serum (Biowhittaker). Cellswere treated with TNF-gamma-beta (aa72-251, 100 ng/ml) in the presenceor absence of cycloheximide (10 micrograms/ml). Caspase activity wasmeasured directly in the wells by adding equal volume of a lysis buffercontaining 25 μM DEVD-rodanine 10 (Roche Molecular Biochemicals), andallowed the reaction to proceed at 37 C for 1 to 2 hours. Release ofrodamine 110 was monitored with a Wallac Victor2 fluorescence platereader with excitation filter 485 nm and emission filter 535 nm.

For the inhibition studies using Fc-proteins or antibodies, theindicated amount of each protein was mixed with either medium or 100ng/ml of TNF-gamma-beta in the presence or absence of cycloheximide. Thereagents were incubated for 1 hour at RT to allow the formation ofprotein-TNF-gamma-beta complexes and then added to the cells. Caspaseactivity was measured as described above.

Murine Graft-Versus-Host Reaction

The F1 (CB6F1) of C57BL/6×BALB/c mice (H-2^(b×d)) were transfusedintravenously with 1.5×10⁸ spleen cells from C57BL/6 mice (H-2^(b)) onday 0. Recombinant TNF-gamma-beta (aa 72-251) protein or buffer alonewas administered intravenously daily for 5 days at 3 mg/kg/day startingon the same day as the transfusion. The spleens of the recipient F1 micewere harvested on day 5, weighed and single cell suspensions preparedfor in vitro assays.

Ex-vivo Mouse Splenocyte Alamar Blue and Cytokine Assays

Splenocytes from normal and the transfused F1 mice were cultured intriplicate in 96-well flat-bottomed plates (4×10⁵ cells/200microliters/well) for 24 days. After removing 100 microliters ofsupernatant per well on the day of harvest, 10 microliters Alamar Blue(Biosource) was added to each well and the cells cultured for additional4 h. The cell number in each well was assessed according to OD₅₉₀ nmminus OD_(590 nm) background, using a CytoFluor apparatus (PerSeptiveBiosystems). Cytokines in the culture supernatant were measured withcommercial ELISA kits from Endogen or R & D Systems followingmanufacturer's instructions.

Example 26 Refolding of TNFR-6 alpha from Inclusion Bodies

Materials and Methods

Reagents were of analytical grade and, unless stated otherwise in theprotocol, purchased from Merck Eurolab. L-arginine was obtained fromAjinimoto Inc, kanamycin from Sigma, lysozyme from Sigma, Alamar Bluefrom Biosource and FasL-FALG from Alexis. Water was filtrated with aMilli-Q system (Millipore).

Protein marker: LMW-marker (Pharmacia, 17-0615-01), stock solution:

Molecular Concentration Protein Weight (kDa) (ug/mL) phosphorylase b97.0 67 albumin 66.0 83 Ovalbumin 45.0 147 Carboanhydrase 30.0 83Trypsin inhibitor 20.1 80 Alpha-lactalbumin 14.4 116MethodsSDS-PAGE

The method of Laemmli (1970) was used as the basis for SDS-PAGEs.Concentration of acrylamide was always 15%. Every protein sample wasboiled at 95 C for 5 min after addition of SDS-sample buffer andsubsequently centrifugated for 5 min at 13,000 rpm (Centrifuge: BiofugePico, Heraeus). SDS-PAGE gels ran 70 min at 150 V in a Mini-Proteansystem (BioRad). Silver staining of SDS gels was done according to theprotocol of Nesterenko et al. J. Biochem. Biophys. Meth. 28 (1984)239-242).

Buffer Systems:

SDS-sample buffer: 250 mM Tris/HCl, pH 8.0; 40% (v/v) glycerine; 5%(w/v) SDS; 5% (v/v) mercaptoethanol

-   Running buffer: 50 mM Tris/HCl; 19 mM glycine; 0.2% (w/v) SDS-   Lower gel buffer (end concentration): 600 mM Tris/HCl, pH 8.0; 0.8%    (w/v) SDS-   Upper gel buffer (end concentration): 100 mM Tris/HCl, pH 6.8; 0.8%    (w/v) SDS    Methods for Determination of Protein Concentrations-   1. Bio-RAD protein assay (Cat. No. 500-0006) with BSA as a standard.-   2. UV-vis-spectra using the theoretical ε_(280 nm). (23390 M⁻¹ cm⁻¹;    http://www.expasy.ch/cgi-bin/protparam) were carried out on a Cary    300 system (Varian Inc.). An OD₂₈₀ of 0.716 corresponds to a    solution of TNFR-6 alpha amino acid residues 30-300 of SEQ ID NO:2,    hereinafter in this example “TNFR-alpha”) with a concentration of 1    mg/ml.    Bacterial Strains and Growth Media-   BL21 (DE3) purchased from Novagen-   LB 0.5 g NaCl, 0.5 g yeast extract, 1 g tryptone in 1 L water-   LB-Agar LB with 15 g Agar-Agar per L water-   2×YT 17 g tryptone, 10 g yeast extract, 5 g NaCl in 1 L water-   SOC 20 g tryptone, 5 g yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM    MgSO₄, 10 mM MgCl₂, 0.4 g glucose in 1 L water    Mammalian Cells.

Jurkat E6-1 cells (ATCC: TIB-1 52) were used in the apoptosis assay

Experimental Protocol:

Transformation of E. coli BL21 (DE3) and Cultivation

E. coli BL21 (DE3) cells were transformed with the pHE4 vector (ATCCDpeosit Number 209645, described in U.S. Pat. No. 6,194,168) containinga polynucleotide encoding amino acid residues 30-300 of SEQ ID NO:2)using a Bio-Rad GenePulser 11 system (2.5 kV, 200 Q, Cuvette with a 2 mmgap). Cells were immediately transferred to SOC medium and shaken for 40min at 37 degrees C. and 600 rpm (Eppendorf Thermomixer Compact). Theywere subsequently plated on LB-Agar petri dishes containing 50micrograms/ml kanamycin and grown overnight at 37 degrees C. A singlecolony was used for an overnight culture and grown in 175 ml LB mediumcontaining 50 micrograms/ml kanamycin at 37 degrees C. and 200 rpm for14 h. 4×5 L erlenmeyer flasks containing 1.5 L 2×YT medium with 50micrograms/ml kanamycin were inoculated with 30 ml overnight cultureeach and grown at 37 degrees C. and 200 rpm for 4 h (until the OD₆₀₀reached 1). Afterwards, cells were induced by addition of 3 mM IPTG andcultivated as before for 3 h more. Harvest was done using a BeckmanAvanti J-20 centrifuge and a JLA 8.1000 rotor at 5000 g and 4 degrees C.for 10 min. Cell pellets were frozen and stored at −20 degrees C.

Preparation of Inclusion Bodies

15 g cells were thawed and homogenized in 75 ml 0.1 M Tris-HCl, pH 7.0,1 mM EDTA using an ultraturrax. After addition of 23 mg lysozyme, thecells were mixed shortly with an ultraturrax and incubated at 4 degreesC. for 30 min. Subsequently 15,000 U benzonase and 3 mM MgCl₂ were addedand the mixture was incubated at 25 degrees C. for 10 min. Cells weredisrupted using a Constant Systems Z-Plus high-pressure homogenizer at1,800 bar (two passages). 0.5 vol. of 67 mM EDTA, 6% Triton X-100, 1.5 MNaCl pH 7.0 were added and the homogenate was incubated at 4 degrees C.for 30 min. Inclusion bodies were sedimented by centrifugation at 4degrees C. and 32,000 g for 10 min (Beckman Avanti J-25 centrifuge; JLA16.250 rotor). Inclusion bodies were resuspended in 120 ml 0.1 MTris-HCl, pH 7.0, 20 mM EDTA using an ultraturrax. The centrifugationstep and the resuspension were repeated 4 times and the resultinginclusion bodies were stored at −20 degrees C. For following analysis ofinclusion bodies in SDS-PAGE a very diminutive amount is sufficient.

Solubilization of TNFR-6 alpha Inclusion Bodies

TNFR-6 alpha inclusion bodies were solubilized by dilution ofapproximately 1 g IBs into 15 ml solubilization buffer (100 mM Tris, pH8.0; 8 M guanidiniumhydrochloride; 100 mM dithiothreitol, 1 mM EDTA) andincubated on a roller shaker at room temperature for 3 h. Aftercentrifugation at 75,000 g (4 degrees C.; 1 h; Beckman centrifuge AvantiJ-25; JA 25.50 rotor) the pH of the supernatant was lowered to 3-4 bydropwise addition of 1 M HCl Two dialysis steps for 2 h at roomtemperature against 4 M guanidiniumhydrochloride, 10 mM HCl usingSpectra/Por dialysis membranes (MWCO 6000-8000 Da; Reorder-No. 132 650)followed by a dialysis against 4 M guanidiniumhydrochloride at 7 degreesC. overnight were carried out to remove dithiothreitol. Proteinconcentration was determined by UV-vis spectroscopy using thetheoretical extinction coefficient of TNFR-6 alpha (see materials andmethods).

Refolding of TNFR-6 alpha

16.7 mg solubilized TNFR-6 alpha (21.6 mg/ml concentration) were addeddropwise (under stirring) to 200 ml refolding buffer (50 mM BICINE, pH9.0, 1 M L-arginine, 0.5 M NaCl, 5 mM oxidized glutathione, 1 mM reducedglutathione) at a temperature of 7 degrees C. This addition was repeatedtwice after 2 and 4 h, respectively. The solution was stirred gentlyovernight (approximately 20 h). After centrifugation at 4 degrees C. and75,000 g (Beckman Avanti J-25 centrifuge, JA 25.50 rotor) for 1 h, thesupernatant was used for buffer exchange.

Buffer Exchange

Buffer exchange took place by applying 60 ml of the refolding samples onan XK 50/20 column packed with 300 ml sephadex G-25 fine (AmershamPharmacia Biotech; Cat. No. 170032-01), equilibrated with elution buffer(50 mM Na₂HPO₄, pH 7.5; 50 mM NaCl). The flow rate was 5 (injection) or10 ml/min (elution a Pharmacia FPLC system at 7 degrees C. At theelution peak of proteins (rise of extinction at 280 nm) 10 fractions of10 ml each were collected and fractions 2-7 pooled. Buffer exchange wasrepeated twice and the fractions containing TNFR-6 alpha were pooled.The protein concentration of the supernatant was determined and sampleswere taken for SDS-PAGE and activity assay.

Further Purification of TNFR-6 alpha using Ion Exchange Chromatography

TNFR-6 alpha fractions from buffer exchange were applied on a 1 mlHiTrap column packed with SP sepharose XL (Amersham Pharmacia Cat.-No.17-5160-01), equilibrated with 50 mM Na₂HP0₄, pH 7.5; 50 mM NaCl. Theflow rate was 0.5 ml/min. Afterwards the column was washed with 20column volumes 50 mM Na2HP04, pH 7.5; 50 mM NaCl. TNFR-6 alpha waseluted by a step-gradient to 50 mM Na₂HP0₄, pH 7.5; 390 mM NaCl andcollected in fractions of 1 ml each. Samples of peak fractions were usedfor determination of protein concentration and SDS/PAGE. Fractionscontaining TNFR-6 alpha were pooled and tested in the activity assay.

Determination of Activity

The determination of refolded TNFR-6-alpha protein activity was assessedusing the in vitro soluble human FasL mediated cytoxicity assay largelyas described in Example 22. A few minor modifications to the assay weremade: Jurkat-E6 cells were used rather than HT-29 cells; the cell numberper well was 10,000 rather than 50,000; the incubation time in thepresence of alamar blue was 56 hours rather than 4 hours; and absorptionmeasurements were carried out ar 620 nm. Concentrations of TNFR-6 alphatested in the assay were 100 ng/ml, 1 microgram/ml and 10 micrograms/ml.

Aliquoting of Samples

Because TNFR-6 alpha tends to aggregate at concentrations above I mg/mlthe sample was diluted to 0.7 mg/mI with 50 mM Na₂HP0₄, pH 7.5; 390 mMNaCl. Samples of 1 ml were aliquoted into 1.5 ml eppendor tubes, frozenin liquid nitrogen and stored at −80 degrees C.

Results of Example 26

Cultivation and Preparation of Inclusion Bodies

From 6 L shake flask culture, 24 grams cells (wet weight) were obtained.These cells yielded approximately 1.5 grams inclusion bodies. Theinclusion body preparation contained about 70% TNFR-6-alpha (residues30-300) (estimation from SDS-PAGE).

Solubilization of TNFR-6 alpha

From 1 grams inclusion bodies approximately 400 mg solubilized proteincould be prepared. In general it is preferrable to have proteinsolubilisate with a high protein content to prevent adding too muchguanidiniumhydrochloride to the refolding reaction. With our procedurewe were able to obtain a solubilisate with 22 mg/ml protein content(estimated with the theoretical extinction coefficient of TNFR-6 alpha).

Refolding of TNFR-6 alpha and Buffer Exchange

Optimal time for refolding was one day. After two days of refolding theyield decreased by approximately 40%. To find the optimal proteinconcentration for the refolding of TNFR-6 alpha, we testedconcentrations ranging from 50-400 micrograms/ml. Although the yield ofsoluble protein was slightly higher at lower concentrations we chose 250micrograms/ml, a concentration that yielded at least 55% soluble proteinafter refolding and avoided working with high refolding volumes. Eventhough only a small aggregation pellet appeared, about 60% of theinitial protein amount were detected by protein determination usingBio-Rad protein assay after centrifugation (refolding yield). The pooledfractions obtained after buffer exchange contained 95% of the appliedprotein amount.

Purification of Refolded TNFR-6 alpha by Ion Exchange Chromatography

Because TNFR-6 alpha inclusion bodies and solubilisate contained a highcontent of other proteins that may interfere with the activity assay weattempted to purify the protein using liquid chromatography. Because ofthe high theoretical pI we have chosen cation exchange chromatography.TNFR-6 alpha bound to SP sepahroseXL in the presence of 50 mM NaCl andcould be eluted with 390 mM NaCl. The main protein contaminants did notbind to this material or eluted at a higher concentration of NaCl.TNFR-6 alpha could be purified to at least 90% (estimated from a silverstained SDS-PAGE). Typical fractions contained 0.5-1.5 mg/ml TNFR-6alpha. At concentrations above 1 mg/ml, the solution became sometimesturbid indicating aggregation of TNFR-6 alpha. We therefore diluted thepooled samples to a concentration below 1 mg/ml. To avoid aggregation werecommend to use a linear gradient to keep the potein concentrationduring elution low. Fractions eluted by a 100% step of high saltcontained TNFR-6 alpha only at approximately 50%.

Because of aggregation, the yield of this purification procedure wasless than 50% of the applied TNFR-6 alpha. So the overall yield of thisstep-gradient procedure, referred to the TNFR-6 alpha content, is20%(Table IX).

TABLE IX Calculation of yield of the used refolding and purificationsteps Estmated purity of TNFR-6 Overall yield Applied alpha (fromSDS-PAGE) Yield (referred to (referred to TNFR-6 Step protein beforeafter total protein content) alpha) Refolding 50 mg 70% 70%  30 mg 60%60% Buffer exchange 28 mg 70% 70%  27 mg 95% 57% Ion exchange 17 mg70% >90%   4.6 mg 27% 20% chromatography

Pooled fractions of purified TNFR-6 alpha were tested for activityimmediately or frozen in liquid nitrogen, stored at −80degrees C. andtested after 5 days in the activity assay.

Activity of the Refolded and Purified Samples

We used the determination of the absorption at 620 nm for the activityassay. With viable cells (without FasL-FLAG) the absorption was around0.4 and with apoptotic cells (with FasL FLAG without TNFR-6 alpha) theabsorption rose to 0.7. The refolded samples of TNFR-6 alpha and thepositive control showed activity in a range from 1-10 micrograms/ml, butnot below (e.g., at 100 ng/ml). The further purified material fromrefolding showed a higher activity than samples after buffer exchangewithout further purification. This may be due to the lower purity of thesamples from buffer exchange, that only contain approximately 70% TNFR-6alpha. But it clearly shows that active (and not just soluble) TNFR-6alpha can b eobtained by refolding even at this high refolding yield(approximately 60% refolding yield).

Storage of refolded TNFR-6 alpha at −80 degrees C. has only a slightinfluence on the activity in the apoptosis assay.

Conclusions

This refolding protocol in connection with the purification of TNFR-6alpha by cation exchange chromatography can be used to produce TNFR-6alpha at a mg-scale. From 6 L shake flask culture (24 grams wet cellweight) approximately 70 mg active TNFR-6 alpha with a purity of atleast 90% can be obtained. After refolding and buffer exchange, a yieldof 60%, referred to the employed amount of solubilized protein at thebeginning of refolding.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples is hereby incorporated herein by reference. Inaddition, the sequence listing submitted herewith and the correspondingcomputer readable form are both incorporated herein by reference intheir entireties. The specification and sequence listing of each of thefollowing U.S. applications are herein incorporated by reference intheir entirety: 60/303,224 filed Jul. 6, 2001; 60/252,131 filed Nov. 21,2000; 60/227,598 filed Aug. 25, 2000; Ser. No. 09/518,931 filed Mar. 3,2000; 60/168,235 filed Dec. 1, 1999; 60/146,371 filed Aug. 2, 1999;60/131,964 filed Apr. 30, 1999; 60/131,270 filed Apr. 27, 1999;60/124,092 filed Mar. 12, 1999; 60/121,774 filed Mar. 4, 1999; Ser. No.09/006,352 filed Jan. 13, 1998 and 60/035,496 filed Jan. 14, 1997.

1. An isolated antibody or fragment thereof that specifically binds to a protein consisting of amino acid residues 31 to 300 of SEQ ID NO: 2 wherein said antibody or fragment thereof decreases the binding of said protein to a TNF family ligand.
 2. The antibody or fragment thereof of claim 1 wherein said TNF family ligand is Fas ligand.
 3. The antibody of claim 1 which antagonizes TNFR-6α mediated inhibition of apoptosis.
 4. The antibody or fragment thereof of claim 1 which is polyclonal.
 5. The antibody or fragment thereof of claim 1 which is monoclonal.
 6. The antibody or fragment thereof of claim 1 which is chimeric.
 7. The antibody or fragment thereof of claim 1 which is a Fab fragment or an F(ab')2 fragment.
 8. The antibody or fragment thereof of claim 1 wherein said antibody or fragment thereof is expressed in a recombinant host cell selected from the group consisting of a CHO cell, yeast cell and E. coli.
 9. An isolated antibody or fragment thereof that specifically binds to a protein consisting of amino acid residues 31 to 300 of SEQ ID NO: 2 wherein said antibody or fragment thereof is polyclonal. 